Effects of epigallocatechin-3-gallate (EGCG) on A549 lung cancer tumor growth and angiogenesis.

Epigallocatechin 3-gallate (EGCG) has cytotoxic effects in many cancer cells. It has been reported that A549 lung cancer cells are markedly resistant to cell death induced by EGCG. In the present study, the effects of EGCG on A549 lung cancer cell growth and angiogenesis were studied. We found that EGCG dose-dependently suppressed A549 cell growth, while A549 cells were markedly resistant to cell death in vitro. Next we found that EGCG increased endostatin expression and suppressed vascular endothelial growth factor (VEGF) expression. We further studied to determine whether EGCG would suppress A549 tumor growth in nude mouse and angiogenesis. EGCG in drinking water significantly suppressed A549 tumor growth in nude mice. Histological analysis revealed that the number of CD34 positive vessels had a tendency to decrease in the tumor. In sum, EGCG had anti-proliferative effects of A549 on tumor growth and showed a tendency to suppress angiogenesis.

Rapid downregulation of cyclin D1 induced by geranylgeranoic acid in human hepatoma cells.

Geranylgeranoic acid (GGA) and its derivatives are currently under development as chemopreventive agents against second primary hepatoma in Japan. We aimed to evaluate chemoprevention targets of GGA and a surrogate marker of chemopreventive response to clarify the molecular mechanism of hepatoma chemoprevention with GGA. Human hepatoma-derived cell lines such as HuH-7, PLC/PRF/5, and HepG-2, were treated with GGA and its derivatives. Cellular dynamics of several cell-cycle-related proteins were assessed by either immunoblotting or immunofluorescence method. The cellular expression of cyclin D1 protein was suppressed immediately after GGA treatment. This reduction was partially blocked by pretreatment with 26S proteasome inhibitor MG-132, indicating that proteasomal degradation was involved in GGA-induced disappearance of cyclin D1. A phosphorylation of retinoblastoma protein (RB) at serine 780, a target site of cyclin D1-dependent kinase 4, was rapidly decreased in GGA-treated HuH-7 cells. Furthermore, subcellular fractionation, Western blotting, and immunofluorescence revealed GGA-induced nuclear accumulation of RB. These results strongly suggest that cyclin D1 may be a target of chemopreventive GGA in human hepatoma cells. GGA-induced rapid repression of cyclin D1, and a consequent dephosphorylation and nuclear translocation of RB, may influence cell cycle progression and may be relevant to GGA-induced cell death mechanisms.

Polished rice as natural sources of cancer-preventing geranylgeranoic acid.

Geranylgeranoic acid, a 20-carbon polyprenoic acid (all-trans 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecatetraenoic acid) and its derivatives were previously developed as synthetic "acyclic retinoids" for cancer chemoprevention. Recently, we demonstrated the natural occurrence of geranylgeranoic acid in various medicinal herbs (Shidoji and Ogawa, 2004). In this present study, we present several lines of evidence to demonstrate that geranylgeranyl diphosphate taken in foods could be metabolized to GGA through geranylgeraniol and geranylgeranyl aldehyde via the following steps: 1) The conversion from geranylgeranyl diphosphate to geranylgeraniol was demonstrated to occur by the action of bovine intestinal alkaline phosphatase, with a K(m) of 46.1 µM. 2) Geranylgeraniol oxidase-mediated conversion of geranylgeraniol to geranylgeranyl aldehyde was revealed in rat liver homogenates, which activity was mainly localized in the mitochondrial fraction. The mitochondrial enzyme showed a K(m) of 92.9 µM. 3) The conversion of geranylgeranyl aldehyde to geranylgeranoic acid by geranylgeranyl aldehyde dehydrogenase in rat liver homogenates was absolutely dependent on exogenously added NAD(+) or NADP(+). The K(m) of the mitochondrial geranylgeranyl aldehyde dehydrogenase was 27.5 µM for geranylgeranyl aldehyde. Taken together, our data suggest that cancer preventive geranylgeranoic acid could be a physiological metabolite from commonly consumed foods.

Downregulation of trefoil factor 3 gene expression in the colon of the senescence-accelerated mouse (SAM)-P6 revealed by oligonucleotide microarray analysis.

Global comparison of the colonic gene expression profiles between 14-month-old senescenceaccelerated mouse (SAM)-P6 mice and SAM-R1 mice, a wild-type control, was conducted with an oligonucleotide microarray containing more than 5,000 mouse genes. Eight genes were upregulated more than two-fold and 94 genes were downregulated more than two-fold in SAM-P6 mice. The three cell defense genes intelectin1 (Itln1), trefoil factor 3 (intestinal) (Tff3) and "deleted in malignant brain tumors 1" (Dmbt1) were among those extensively downregulated. Quantitative RT-PCR analysis confirmed that Itln1 mRNA was almost undetectable in SAM-P6 colon, whereas it was readily detected in SAM-R1 colon. Colonic expression of both Tff3 and Dmbt1 mRNA was also substantially decreased, to one third and two thirds of the levels in SAM-R1 mice, respectively. A 14 kDa Tff3 dimer was detected by Western blotting in the colon of all three SAM-R1 mice, but was not present in three SAM-P6 mice. No upregulation of 3 cell defense genes was detected in 3-month-old SAM-R1 as well as SAM-P6 mice. These results suggest that a diminution of the intestinal trefoil factor system may be involved in the acceleration of aging in SAM-P6 mice.

Prohibitin protects against hypoxia-induced H9c2 cardiomyocyte cell death.

We recently demonstrated that short time exposure to hypoxia (15 min) in H9c2 cardiomyocytes protected cells against cell death, and longer exposure to hypoxia induced cell death. To understand the molecular mechanism concerning cell death and survival, it is intriguing to identify survival factors against cell death. Using proteomics analysis, levels of proteins derived from H9c2 cells exposed to hypoxia and normoxia were compared and candidates for survival factor were identified. One of the candidates was a prohibitin. Overexpression of prohibitin inhibited H9c2 cell death induced by hypoxia for longer hours. We further clarified the mechanism of cell death. Overexpression of prohibitin inhibited decrease of mitochondrial membrane potential levels, decrease of Bcl-2 level in mitochondria and cytochrome c release to cytosol from mitochondria induced by hypoxia. The mechanism for survival was that overexpression of prohibitin inhibited cytochrome c release by decrease of mitochondrial membrane potential levels and decrease of Bcl-2 level. Taken together, identified prohibitin may function as a survival factor against hypoxiainduced cell death.

Enhanced Glucose Requirement in Human Hepatoma-derived HuH-7 Cells by Forced Expression of the bcl-2 Gene.

To explore the metabolic effects of Bcl-2 in tumor cells, a stable clone of HuH-7/bcl-2 and its control HuH-7/neo were established. Mitochondrial localization of ectopic Bcl-2 was demonstrated both by western blotting and immunofluorescence. HuH-7/bcl-2 cells consumed glucose at a higher rate, exhausted the available cellular ATP and died on day 9, while HuH-7/neo cells were still alive for 10 days under the same condition where cells were cultured without replenishment of the medium. The expression of the hexokinase II gene was up-regulated in HuH-7/bcl-2 at its protein level. Taken together, we suggest that the forced expression of Bcl-2 in human hepatoma may cause the cells to become more glucose-dependent for survival.

Expression of the hepatic endothelin system in human cirrhotic livers.

It is considered that endothelin-1 participates in the development of liver cirrhosis and it has been recognized that every component of the endothelin system is upregulated in cirrhotic livers. However, the expression pattern of this system, including interaction between its components, is not fully understood in human livers. In this study, the expression pattern of the endothelin system was examined. Immunohistochemical analysis for endothelin-1, endothelin receptors and endothelin-converting enzyme was performed in 16 cirrhotic and 17 normal human liver tissues. Peptides, proteins, and RNAs extracted from the livers were also investigated using quantitative assays for the components of the hepatic endothelin system. Hepatic endothelin-1 levels were significantly higher in cirrhotic livers (0.084 +/- 0.052 pg/mg wet liver) than in normal livers (0.041 +/- 0.032 pg/mg; p < 0.01), and were closely related to the severity of liver fibrosis and portal hypertension. Immunoreactivity for endothelin-1, endothelin receptors, and endothelin-converting enzyme was detected mainly in fibrous areas and in the hepatic vasculature, and was enhanced in cirrhosis. Although there was a negative correlation between the expression of receptor mRNA and the hepatic endothelin-1 level, the amounts of the mRNAs were greater in cirrhotic livers than in normal livers. However, expression of endothelin-converting enzyme in cirrhotic livers was increased at the protein level but was relatively reduced at the mRNA level. These findings suggest that the hepatic endothelin system is activated in human cirrhotic livers in association with worsening of the disease, but that the regulation of the components of this system in this disorder is complex.