Tadayoshi Hasuma

Growth-Inhibitory Effect of a High Glucose Concentration on Osteoblast-like Cells

Impaired bone formation resulting from a decline of osteoblast activity has been implicated in the pathogenesis of diabetic osteopenia. We examined the effects of high glucose concentration alone, independent of insulin deficiency, on the growth of a human osteoblast-like cell line (MG-63). Sustained exposure to high glucose for 7 days inhibited cell growth in a concentration-dependent manner up to 49.5 mmol/L, as compared with cells cultured with a normal glucose concentration (5.5 mmol/L) or a high mannitol concentration (an iso-osmolar control). Glucose (49.5 mmol/L) attenuated the increment either in DNA content or in [3H]thymidine incorporation induced by insulin-like growth factor I (IGF-I). The IGF-I-induced increase of ornithine decarboxylase (ODC) activity, which plays an important role in cell growth, was also attenuated. The half-life of ODC protein was not shortened by the high glucose culture, but the intracellular content of putrescine (an end product of ODC) was significantly decreased. These changes did not occur in the high mannitol culture, strongly suggesting a specific effect of glucose. In summary, our observations suggest that a high glucose concentration significantly impairs the proliferative response of osteoblastic cells to IGF-I and that the defective cell function caused by sustained exposure to high glucose levels might contribute to impaired bone formation in patients with diabetic osteopenia.

Development of hepatocytes from ES cells after transfection with the HNF-3beta gene.

We have attempted to generate embryonic stem (ES) cell‐derived hepatocytes expressing liver‐specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)‐3β‐transfected ES cells are cultured in α‐MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)‐2 in the three‐dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF‐3β‐transfected ES cells also had hepatocyte‐like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.

Mechanism of the Anti-Cancer Activity of Zizyphus jujuba in HepG2 Cells

The Zizyphus jujuba fruit has been used as a traditional Chinese medicinal herb and considered to affect various physiological functions in the body for thousands of years. However, its anti-cancer activity and mechanism of action remain to be elucidated. We investigated the anti-cancer activity of Zizyphus jujuba Mill and its underlining mechanisms of action in human hepatoma cells (HepG2) and found that the extract of Z. jujuba decreased the viability of the cells. Further extraction of the initial Z. jujuba extract with organic solvents revealed that the chloroform fraction (CHCl(3)-F) was the most effective. Interestingly, the CHCl(3)-F induced not only apoptosis but also G1 arrest at a low concentration (100 mug/ml) and G2/M arrest at a higher concentration (200 mug/ml) by cell cycle assay. Apoptosis, an increase in intracellular ROS (reactive oxygen species) level, a decline of mitochondrial membrane potential at low Z. jujuba concentrations, and a ROS-independent mitochondrial dysfunction pathway at high concentrations were all observed. CHCl(3)-F-induced G1 arrest in HepG2 cells was associated with an increase in hypohosphorylation of Rb and p27(Kip1), and a decrease of phosphorylated Rb. However, CHCl(3)-F-induced G2/M arrest in HepG2 cells correlated with a decrease of the p27(Kip1) levels and generation of the phosphorylation of p27(Kip1), however the hypohosphorylation of Rb protein remained. Collectively, our findings suggest that the CHCl(3)-F extract of Z. jujuba extract induced a concentration dependent effect on apoptosis and a differential cell cycle arrest in HepG2 cells.

Transforming Growth Factor-β and Hepatocyte Growth Factor Produced by Gastric Fibroblasts Stimulate the Invasiveness of Scirrhous Gastric Cancer Cells

Scirrhous gastric carcinoma is characterized by cancer cells that infiltrate rapidly in the stroma with extensive growth of fibroblasts. In the present study, we examined the effect of gastric fibroblasts on the invasiveness of a Scirrhous gastric cancer cell line, OCUM‐2D, using an invasion assay. Gastric fibroblast‐derived conditioned medium (CM) significantly stimulated the invasiveness of OCUM‐2D cells, as did transforming growth factor‐β (TGF‐β) and hepatocyte growth factor (HGF). The stimulating activity of gastric fibroblast‐derived CM was inhibited significantly by anti‐TGF‐β neutralizing antibody or anti‐HGF neutralizing antibody. TGF‐β and HGF were detected in the gastric fibroblast‐derived CM, and TGF‐β receptor and C‐met (HGF receptor) were expressed on OCUM‐2D cells. Thus, TGF‐β and HGF produced by gastric fibroblasts appear to affect the invasiveness of scirrhous gastric cancer cells. TGF‐β was also detected in the conditioned medium derived from OCUM‐2D cells, though HGF was not. TGF‐β appears to affect the invasiveness of OCUM‐2D cells in both paracrine and autocrine fashions.

Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro

Green tea extract and its polyphenolic components have been found to possess anticarcinogenic, antimutagenic, antihypertensive and antihepatotoxic effects, and several mechanisms have been proposed for these effects. In this study, the effects of five tea polyphenols, (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-) epicatechin-3-gallate (ECG), ( -) epicatechin (EC) and (+)-catechin (C), were examined on the viability of Ehrlich ascites tumor cells in vitro and a possible relationship with tyrosine phosphorylation was determined. Proteins extracted from the cells treated with the tea polyphenols were separated by SDS-PAGE, and tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine antibody and the extent of phosphorylation determined. EGC (100 microM) caused a significant decrease in cell viability to 4.1 +/- 0.2% of the control value, and this correlated with a stimulation of protein tyrosine kinase (PTK) activity. EGCG (100 microM) also caused a slight decrease in cell viability (approximately 70% of the control value) but this and the other polyphenols, which had no effect on cell viability likewise, had no effect on tyrosine phosphorylation. Tyrosine phosphorylations of 42 and 45 kDa proteins were also observed for EGC. Further evaluation of the effect of EGC showed that the activity of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis in cells, decreased significantly as well. A significant correlation has therefore been observed between a cellular event, namely, a reduction in the viability of Ehrlich ascites tumor cells and an association with a tyrosine phosphorylation of 42 and 45 kDa proteins by the polyphenol EGC.

Growth inhibitory effect of green tea extract and (-)-epigallocatechin in Ehrlich ascites tumor cells involves a cellular thiol-dependent activation of mitogenic-activated protein kinases.

The effect of green tea extract (GTE) in Ehrlich ascites tumor cells (EATC) was studied with respect to changes in the intracellular kinase system involving mitogen-activated protein kinases (MAPKs) and cellular thiol. We have previously shown a reduction in viability of EATC and tyrosine phosphorylation of 42 and 45 kDa proteins by GTE and its polyphenolic component, Epigallocatechin (EGC) (D.O. Kennedy, S. Nishimura, T. Hasuma, Y. Yoshihisa, S. Otani, I. Matsui-Yuasa, Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro, Chem. Biol. Interact. 110 (1998) 159-172). Furthermore, GTE and EGC significantly decreased both cellular non-protein and protein sulfhydryl levels in EATC, but replenishing thiol stores with N-acetylcysteine (NAC) caused a recovery in cell viability, and therefore SH groups were identified as a novel target of green tea cytotoxicity (D.O. Kennedy, M. Matsumoto, A. Kojima, I. Matsui-Yuasa, Cellular thiol status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells, Chem. Biol. Interact. 122 (1999) 59-71). In this study, we have observed the stimulation of three forms of MAPK, namely ERK1/2, JNK/SAPK and p38, by EGC, which were dose and time-dependent. These MAPK stimulations were found to be cellular thiol status-dependent events as NAC reversed these stimulations. Furthermore, inhibition of the p38 MAPK pathway using the p38 inhibitor SB203580 caused a marked dose-dependent reduction in the decrease in cell viability caused by EGC treatment. Inhibiting the Erk1/2 MAPK pathway using the MEK inhibitor PD098059 caused a slight change in the decrease in cell viability by EGC. These may suggest that the cytotoxicity associated with EGC was more associated with the other MAPKs than with ERK1/2. This may be the first study of its kind providing a novel evidence of a role for different forms of MAPKs in the antitumor effect of green tea polyphenols, especially EGC, in Ehrlich ascites tumor cells.

Overexpression of cyclin D1 in rat esophageal carcinogenesis model

Overexpression of cyclin D1 in human esophageal carcinomas has been well documented. The aim of the present study was to assess the expression of cyclin D1 in different types of esophageal epithelial lesions induced by N‐nitrosomethylbenzylamine (NMBA) in rats. A total of 30 rats received s.c.‐injections, five times/week, of 1.0 mg/kg NMBA for a period of 5 weeks followed by the same dose once per week for another 10 weeks. An additional 15 rats were given saline and used as controls to provide normal epithelium. The tumor incidence was 100% at the termination point of 21 weeks. Seventeen rats (57%) showed nuclear staining for cyclin D1, with a great variation in the intensity, as demonstrated by using an immunohistochemical technique. The cyclin D1 positive indices were in the range of 0% to 60% of the individual cells. Negligible staining was observed for normal esophageal epithelium, with a minimal increase in hyperplastic and dysplastic lesions. A significant elevation of cyclin D1 levels was observed in tumors. However, no significant differences were found between papillomas and carcinomas. The immunohistochemical results were confirmed by western blotting analysis. Tumors, papillomas and carcinomas overexpressing cyclin D1 had elevated proliferating cell nuclear antigen (PCNA) indices (P<0.05). The correlation coefficient of overexpressions of PCNA and cyclin D1 was r=0.7 for papillomas, but only r=0.3 for carcinomas. The study thus provides strong evidence of relatively early Overexpression of cyclin D1 during tumorigenesis in the present rat esophageal model. Cyclin D1 expression is not simply a direct consequence of increase cell proliferation.

Dopamine as a novel antimigration and antiproliferative factor of vascular smooth muscle cells through dopamine D1-like receptors

Vascular smooth muscle cell (VSMC) migration and proliferation are believed to play key roles in atherosclerosis. To elucidate the role of vascular dopamine D1-like receptors in atherosclerosis, the effects of dopamine and specific D1-like agonists SKF 38,393 and YM 435 on platelet-derived growth factor (PDGF) BB-mediated VSMC migration and proliferation were studied. We observed that cells stimulated by PDGF-BB (5 ng/mL), showed increased migration and proliferation. These effects were prevented by coincubation with dopamine, SKF 38,393, or YM 435 (1 to 10 mumol/L), and this prevention was reversed by Sch 23,390 (1 to 10 mumol/l), a specific D1-like antagonist. These actions are mimicked by forskolin (1 to 10 mumol/L), a direct activator of adenylate cyclase and 8-bromo-cAMP at 0.1 to 1 mmol/L and are blocked by a specific protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H 89), but not blocked by its negative control, N-[2-(N-formyl)-p-chlorociannamylamino)ethyl]-5-isoquinoline sulfonamide (H 85). PDGF-BB (5 ng/mL)-mediated activation of phospholipase D, protein kinase C, and mitogen activated protein kinase activity were significantly suppressed by coincubation with dopamine. These results suggest that vascular D1-like receptor agonists inhibit migration and proliferation of VSMC, possibly through protein kinase A activation and suppression of activated phospholipase D, protein kinase C, and mitogen-activated protein kinase activity.

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of retinoblastoma protein phosphorylation by (-)-epigallocatechin.

Tea polyphenols have been shown to inhibit tumor cell growth, but there is limited information on their effects on cell signaling and cell cycle control pathways. We have shown the involvement of such mechanisms as activation of mitogenic activated protein kinases, decreases in ornithine decarboxylase activity and in cellular thiol levels, elicitation of mitochondrial cytochrome c release, and activation of caspases by the green tea galloyl polyphenol, epigallocatechin (EGC). In the current study, we sought to determine how EGC alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells. The significant finding here is that EGC caused a dose-dependent accumulation of cells in the G1 phase and a decrease in the phosphorylation of the retinoblastoma (Rb) protein, which was also in a cellular thiol-dependent manner. The involvement of a cellular thiol-dependent modulation in Rb phosphorylation leading to the regulation of tumor cell growth by a green tea polyphenol is a novel observation, to the best of our knowledge.

Growth inhibitory effect of green tea extract in Ehrlich ascites tumor cells involves cytochrome c release and caspase activation

We reported previously that the mechanism by which Green tea extract (GTE) elicited growth-inhibitory effects in Ehrlich ascites tumor cells involved a decrease in ornithine decarboxylase (ODC) activity and in cell viability. Decrease in ODC activity has been associated with apoptotic cell death and we therefore studied changes in cytochrome c release and caspase activation, which characterize apoptosis. GTE caused a dose- and time-dependent increase in caspase-3-like protease activation, preceded by a release of cytochrome c from the mitochondria. Inhibiting the activation of caspase-3 with acetyl-Asp-Glu-Val-Asp-alpha-aldehyde (caspase inhibitor) caused a reversal in the effect on cell viability.