Yoshizumi Matsukawa

Butyrate Activates the WAF1/Cip1 Gene Promoter through Sp1 Sites in a p53-negative Human Colon Cancer Cell Line

Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mm butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at −82 and −69 relative to the transcription start site. We also found that a TATA element at −46 and two overlapping consensus Sp1 sites at −60 and −55 are essential for the basal promoter activity ofWAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion.

Promoter activation and following induction of the p21/WAF1 gene by flavone is involved in G1 phase arrest in A549 lung adenocarcinoma cells

Flavonoids are present in many plants including edible fruits and vegetables. Recently, many of the biological activities of flavonoids have been elucidated. Flavone is a well known flavonoid, and many of its derivatives have been shown to have anti‐proliferative effects on several cancer cells. We report here that flavone can effectively inhibit the cell growth of human lung adenocarcinoma A549 cells in a dose‐dependent manner, and 100 μM flavone causes cell cycle arrest at the G1 phase. As a mechanism underlying the cell cycle arrest, flavone markedly increases the mRNA and protein levels of a universal inhibitor of cyclin‐dependent kinase, p21/WAF1, and inhibits phosphorylation of retinoblastoma (RB) protein. Although A549 cells possess wild‐type p53, flavone does not induce the p53 protein, suggesting that p21/WAF1 induction is p53‐independent. In addition, 100 μM flavone significantly increases the promoter activity of the p21/WAF1 gene by 5‐fold. These results suggest that the G1 phase arrest by flavone is due to p53‐independent transcriptional induction of the p21/WAF1 gene and the subsequent dephosphorylation of RB protein.

Effects of Quercetin and/or Restraint Stress on Formation of Aberrant Crypt Foci Induced by Azoxymethane in Rat Colons

The present study examines the effect of dietary quercetin and/or restraint stress on the formation of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in the colon. Female F344 rats were divided into four groups. All animals were given intraperitoneal injections of AOM. The animals were fed a basal diet (group A, C), or a 2% quercetin-supplemented diet (group B, D). The animals were put individually to narrow wire cages for 1 h every day throughout the study to expose them to mild restraint stress (group C, D). At week 5, all the rats were killed and analyzed for ACF in the colon. The number of ACF increased significantly in the animals subjected to stress (p < 0.05). On the other hand, dietary quercetin significantly reduced the number of ACF in both the nonstress (p < 0.001) and stress groups (p < 0.05). These findings suggest that quercetin might have a potential as a chemopreventive drug for colon cancer despite stress.

New isoflavones from Belamcandae Rhizoma

Four new isoflavones (iristectrigenin A-7-glucoside (2), 8-hydroxytectrigenin (5), 8-hydroxyiristectrigenin A (6), and 8-hydroxyirigenin (7–1) have been isolated from Belamcandae Rhizoma (the rhizome of Belamcanda chinensis), along with nine known compounds: tectridin (1), tectrigenin-4′-glucoside (3), astragalin (4), irilin D (7–2), isotectrigenin (8), tectrigenin (9), iristectrigenin B (10), hispiludin (11–1), and irigenin (11–2). The structures of 2, 5, 6 and 7–1 were determined by spectroscopic methods, including high-resolution mass spectrometry (HR-MS), proton nuclear magnetic resonance (1H-NMR), carbon-13 NMR (13C-NMR), and various two-dimensional (2D)-NMR techniques.

Ammonia Inhibits Proliferation and Cell Cycle Progression at S-Phase in Human Gastric Cells

Helicobacter pylori (Hp) has strong ureaseactivity and produces a large amount of ammonia in thestomach. In animal studies, ammonia was shown toaccelerate cell kinetics of gastric mucosa, andlong-term exposure of the stomach to ammonia leads tomucosal atrophy. To understand this process, we examinedthe effects of ammonia on the growth and cell cycleprogression of human gastric cancer cell lines (HGC-27, MKN1, MKN45) using flowcytometric analysis. Ineach cell line, ammonia inhibited the cell growth in adose-dependent manner and caused significantaccumulation of S-phase cells at a cytostatic dose. DNA synthesis of HGC-27 cells treated with ammoniawas also suppressed to about 50% of that of theuntreated cells. Similar effects were observed onaddition of ammonium chloride at the same concentration, while adjusting the pH of the media with NaOHalone to that with the cytostatic dose of ammonia didnot affect the cell cycle progression. Theseobservations indicate that ammonia induces S-phasearrest in gastric cells independently of pH.

Growth Inhibition of A549 Human Lung Adenocarcinoma Cells by L‐Canavanine Is Associated with p21/WAF1 Induction

L‐Canavanine (CAV) is a higher plant nonprotein amino acid and a potent L‐arginine antimetabolite. CAV can inhibit the proliferation of tumor cells in vitro and in vivo, but little is known regarding the molecular mechanisms mediating these effects. We demonstrated that the treatment of human lung adenocarcinoma A549 cells with CAV caused growth inhibition; G1 phase arrest is accompanied by accumulation of an incompletely phosphorylated form of the retinoblastoma protein, whose phosphorylation is necessary for cell cycle progression from G1 to S phase. In addition, CAV induces the expression of p53 and subsequent expression of a cyclin‐dependent kinase inhibitor, p21/WAF1. The p53–dependent induction of p21/WAF1 and the following dephosphorylation of the retinoblastoma protein by CAV could account for the observed CAV‐mediated G1 phase arrest.

Quercetin Enhances Tumorigenicity Induced by N-Ethyl-N'-Nitro-N-Nitrosoguanidine in the Duodenum of Mice*

Quercetin, a flavonoid, widely distributed in many fruits and vegetables, is well known to have an antitumor effect despite its mutagenicity. In this study, we examined the effect of dietary quercetin on duodenum-tumorigenicity of mice induced by a chemical carcinogen, N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Eight-week-old male C57BL/6 mice were divided into 4 groups; ENNG without quercetin (group A), ENNG with 0.2% quercetin (group B), ENNG with 2% quercetin (group C), and 2% quercetin without ENNG (group D). ENNG was given in drinking water for the first 4 weeks, and thereafter quercetin was given in a mixed diet. At week 20, the average number of duodenal tumors per mouse was significantly higher in group C (mean±SE, 7.26±1.75, p<0.05) than in group A (2.32±0.31). The size of the duodenal tumors increased significantly in group B (1.79±0.09 mm, p<0.001) compared with group A (1.43±0.09 mm). In contrast, no duodenal tumor was induced in group D. The present findings suggest that excessive intake of quercetin occasionally is a risk factor for carcinogenesis of some specific organs such as the upper intestine.

Effects of Flavonoids on Cell Cycle and Differentiation in Cancer Cells

Flavonoids are plant pigments found in edible fruits and vegetables. We examined the effects of Flavonoids on cell cycle progression and differentiation of malignant tumor cells. Flow cytometric analysis showed that flavone, luteolin, quercetin, and daidzein induced G1 arrest, whereas genistein specifically inhibited the cell cycle at G2-M in the human gastric cancer cell line HGC-27. Other flavonoid, apigenin, also arrested the cell cycle of B104 rat neuronal cells at G2-M phase. Inhibition of the cell cycle at two major checkpoints, G 1 and G2-M, may be important to the antitumor activities of the flavonoids. Furthermore, apigenin induced morphological differentiation of B104 cells, which suggests an unknown pathway of differentiation from G2-M.

Three Cases of Panic Disorder Successfully Treated with Kampo Formulae

小半夏加茯苓湯の方意を含む漢方薬治療で症状の著明改善が図れたパニック障害の3例を経験した。症例1：47歳男性，運転士，血圧上昇を伴う一過性脳虚血発作を引き金に発症。身体疾患の発症がストレスを増強させ心下の気水が鬱して痰熱となりパニック発作になったと考えられる。小半夏加茯苓湯に黄連湯の方意をあわせて症状は消失した。症例2：49歳女性，主婦，家庭内のストレスをきっかけに発症。肝血不足，疎泄不良と脾虚が重なり心下の飲を起こしたと考え，小半夏加茯苓湯を含む茯苓飲合半夏厚朴湯エキスに加味逍遙散エキスを合わせて奏効した。症例3：32歳女性，主婦，子育ての疲労をきっかけに発症。疲れによって脾虚から心下の飲がおこるとともに血虚に陥ったと考え，小半夏加茯苓湯と十全大補湯合方を用い奏効した。『金匱要略』の方剤小半夏加茯苓湯は，中焦の飲と気の上逆を引き降ろすことで「心下痞」「眩悸，」を全例で改善し，症例毎に随証治療をあわせおこなうことでパニック発作が消失した。