Effect of promoter methylation of the p16 gene on phosphorylation of retinoblastoma gene product and growth of hepatocellular carcinoma cells.

The biological significance of hypermethylation of p16 gene promoter in human hepatocellular carcinoma (HCC) cells remains to resolved. In order to clarify the significance of methylation of p16 gene promoter, we examined the methylation status of p16 gene in association with phosphorylation of retinoblastoma gene product (pRb) and cell growth in human HCC cell lines. The presence of methylation was examined by methylation-specific PCR. Expression and phosphorylation of p16 and pRb were examined by Western blot analysis. Genetic changes were analyzed by multiplex PCR and DNA sequencing. The effect of demethylation of p16 was assessed by cell growth. p16 gene promoter was methylated in HuH7 and HLF cells. The demethylating agent, 5-aza-2-deoxycytidine (5-Aza-CdR), upregulated p16 mRNA in HuH6 and HuH7 cells. 5-Aza-CdR increased p16 protein expression in HuH6, HuH7, and HLF cells, and it clearly decreased the phosphorylation level of pRb in HuH6, HuH7 and PLC/PRF/5 cells. Treatment with 5-Aza-CdR inhibited the growth of HuH7 cells. Homozygous deletion and significant mutations were absent. Methylation in the p16 promoter region is biologically significant, being associated with phosphorylation of pRb and cell growth in human HCC cells.     

Improved trends in survival and engraftment after single cord blood transplantation for adult acute myeloid leukemia

Unrelated cord blood transplantation (CBT) is an alternative curative option for adult patients with acute myeloid leukemia (AML) who need allogeneic hematopoietic cell transplantation (HCT) but lack an HLA-matched related or unrelated donor. However, large-scale data are lacking on CBT outcomes for unselected adult AML. To investigate the trends of survival and engraftment after CBT over the past 22 years, we retrospectively evaluated the data of patients with AML in Japan according to the time period of CBT (1998–2007 vs 2008–2013 vs 2014–2019). A total of 5504 patients who received single-unit CBT as first allogeneic HCT for AML were included. Overall survival (OS) at 2 years significantly improved over time. The improved OS among patients in ≥ complete remission (CR)3 and active disease at CBT was mainly due to a reduction of relapse-related mortality, whereas among patients in first or second CR at CBT, this was due mainly to a reduction of non-relapse mortality. The trends of neutrophil engraftment also improved over time. This experience demonstrated that the survival and engraftment rate after CBT for this group has improved over the past 22 years.Subject terms: Acute myeloid leukaemia, Cancer immunotherapy     

Identification of Potential mRNA Biomarkers in Milk Small Extracellular Vesicles of Enzootic Bovine Leukosis Cattle

Enzootic bovine leukosis (EBL) is a disease caused by bovine leukemia virus (BLV); only a small percentage of BLV-infected cattle develop EBL and present with B-cell lymphosarcoma. There is no vaccine against BLV, treatment for EBL, or method for predicting the possibility of EBL onset, thus making EBL control difficult. Herein, to explore biomarkers for EBL in milk, we examined the mRNA profiles of small extracellular vesicles (sEVs) in milk from four BLV-uninfected and four EBL cattle by microarray analysis. It was revealed that 14 mRNAs were encapsulated in significantly higher quantities, and these mRNAs were therefore selected as biomarker candidates. Primers for these mRNAs were designed, and nine primer sets were available for quantitative real-time PCR. Nine mRNAs were evaluated for their availability as biomarkers for EBL using sEVs from newly-collected milk of 7 uninfected and 10 EBL cattle. The quantities of eight mRNAs (TMEM156, SRGN, CXCL8, DEFB4A, FABP5, LAPTM5, LGALS1, and VIM) were significantly higher in milk sEVs of EBL cattle than in those of uninfected cattle. Therefore, our findings indicate that these eight mRNAs in milk sEVs can be used as potential EBL biomarkers with combination use, although single mRNA use is not enough. Consequently, cattle at risk of EBL onset can be identified by monitoring the fluctuation in quantities of these mRNAs in milk before they develop EBL.     

The PRB-dependent FOXO1/IGFBP-1 axis is essential for progestin to inhibit endometrial epithelial growth

Progestin inhibits the growth of normal and cancerous endometria via the progesterone receptor (PR), but the distinct functions and signalings of PR subtypes have not been fully understood. The aim of the present study was to dissect the key pathways of progestin to inhibit endometrial epithelial growth. Immortalized endometrial epithelial cells (EM-E6/E7/TERT) with stable PRA or PRB expression were established and used for the experiments. In vitro growth inhibition by progestin was mainly observed in EM-E6/E7/TERT cells with PRB rather than those with PRA. RT-PCR assay confirmed that FOXO1, a key gene for progestin action, was up-regulated by progestin in a PRB-dependent manner. cDNA microarray analysis identified IGFBP-1, which contains FOXO1 binding sites on its promoter, to be induced by medroxyprogesterone acetate (MPA) in EM-E6/E7/TERT cells with PRB but not with PRA. siRNA knockdown of FOXO1 disturbed the induction of IGFBP-1 by MPA, while IGFBP-1 knockdown showed no effect on MPA-induced FOXO1 expression, indicating that FOXO1 is an upstream regulator of IGFBP-1. Luciferase reporter assays showed that MPA activated the IGFBP-1 promoter, which was cancelled by FOXO1 knockdown. Chromatin immunoprecipitation assay confirmed the in vivo binding of FOXO1 to the core promoter of IGFBP-1. IGFBP-1 knockdown significantly attenuated the growth inhibitory effects of MPA. The FOXO1/IGFBP-1 axis is essential for PRB-dependent growth inhibition of endometrial epithelial cells, offering a potential therapeutic clue to enhance the progestin effect.     

Cyclophosphamide and 5-fluorouracil act synergistically in ovarian clear cell adenocarcinoma cells

Chemosensitivity to the drugs plays a crucial role in the treatment of ovarian cancer. In this study, we evaluate the cytotoxicity of chemotherapeutic agents in six ovarian cancer cell lines; four clear cell adenocarcinoma and two serous papillary adenocarcinoma, using seven single drugs and seven sets of drug combinations with tetrazolium-based semiautomated colorimetric (MTT) assay. The drug concentration which produced 50% growth inhibition (IC50) of cisplatin was within clinically achievable range in five cell lines. The area under the curve (AUC) at IC50 of cyclophosphamide was below the clinically achievable AUC in two serous papillary cell lines. Paclitaxel was more effective in clear cells than serous papillary cells. The intensification of cytotoxicity was observed in the combinations of paclitaxel and cisplatin, and cyclophosphamide and cisplatin or 5-fluorouracil irrespective of histopathological characteristics of the original tumor. Our results indicate that ovarian cancer cell lines respond to chemotherapeutic agents heterogeneously depending upon histopathological features, indicating individualized regimens may improve survival in ovarian cancer patients.     

Phenolic glycosides from berries of Pimenta dioica

Four new phenolic glycosides, (2-hydroxy-3-methoxy-5-allyl)phenyl beta- d-(6-O-E-sinapoyl)glucopyranoside (1), (1' R,5' R)-5-(5-carboxymethyl-2-oxocyclopentyl)-3 Z-pentenyl beta-D-(6-O-galloyl)glucopyranoside (2), (S)-alpha-terpinyl [alpha-L-(2-O-galloyl)arabinofuranosyl]-(1-->6)-beta-D-glucopyranoside (3), and (R)-alpha-terpinyl [alpha-L-(2-O-galloyl)arabinofuranosyl]-(1-->6)-beta-D-glucopyranoside (4), were isolated from the berries of Pimenta dioica together with eight known flavonoids. The structures of 1-4 were elucidated on the basis of MS and NMR data and enzymatic hydrolysis. All four glycosides showed radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals.     

Anti-tumor effects of water-soluble propolis on a mouse sarcoma cell line in vivo and in vitro

The honeybee product propolis and its extracts are known to have biological effects such as antibiotic, anti-viral, anti-inflammatory and anti-tumor activities. This study was designed to investigate whether water-soluble propolis (WSP) inhibits tumor growth. The tumor cell line used was mouse sarcoma 180 (S-180), and its growth was determined in vitro and in vivo with exposure to different concentrations of WSP. The effects of WSP on tumor cells in vitro were evaluated by measuring the intracellular uptake of 3H-thymidine. 3H-thymidine uptake was inhibited in accordance with the concentration of WSP. The minimum concentration of WSP necessary for 3H-thymidine uptake inhibition was 1.0 microg/ml and uptake was suppressed to 88% of the level in non-treated cells at this concentration. In an experiment using tumor-bearing mice, oral administration of WSP was begun 24 hours after transplantation of S-180 cells. WSP was administered to the mice 5 times, every other day for 10 days. The doses were 320 mg/kg (10 mg/mouse) or 960 mg/kg (30 mg/mouse) of body weight. All mice were sacrificed 10 days after transplantation, and tumor growth was evaluated. The orally administered WSP significantly inhibited the growth of transplanted tumors (p < 0.05). Furthermore, histological findings revealed a significant reduction in mitotic cells and tumor invasion of the muscular tissue at both dose-levels of WSP.     

Level of S100B protein expression in the amnion at various gestational ages in the third trimester of normal pregnancies

BACKGROUND: S100B protein is a unique calcium-binding protein. Its biological role within the cell populations is not completely defined. Some pathological conditions that develop during pregnancy could affect S100B concentrations in the amniotic fluid, cord blood, and maternal serum. The aim of our study was to assess the correlation between S100B protein expression in the amnion, amniotic fluid and gestational age in the third trimester of uncomplicated pregnancies. METHODS: Amnion, amniotic fluid, maternal peripheral and umbilical cord blood samples were collected from healthy women who delivered at 31-36 weeks (n=17), 37-40 weeks (n=22), and 41-42 weeks (n=21). The expression of S100B in the amnion was assessed by immunohistochemistry and real-time (RT)-PCR, and its concentrations in amniotic fluid, maternal and cord blood sera were determined by ELISA. RESULTS: The S100B protein expression in the amnion and its concentrations in amniotic fluid, maternal and cord blood sera of patients in the third trimester were not significantly different at various gestational ages. CONCLUSIONS: The S100B protein expression in the amnion and the S100B protein concentrations in amniotic fluid, maternal and cord blood do not vary significantly in the third trimester of uncomplicated pregnancies.     

Trends in dietary fiber intake in Japan over the last century

Summary. Background: Insufficient intake of dietary fiber (DF) is currently a major problem in the overall promotion of health in the general population in Japan. Aim of the study: To analyze the time trends in DF intake, including DF density (total DF intake/1,000 kcal), and the ratio of water-insoluble fiber to water-soluble fiber (IS ratio) in Japan. Methods: The time trend in DF intake in Japan was calculated from data compiled in the Japanese National Nutrition Survey. Results: The mean daily DF intake (total DF intake) in 1952 was 20.5 g/day, which rapidly declined to about 70 % of the 1952 level in 1970, after which there was little change to 1998. DF density in 1952 was 9.7 g/1000 kcal, which declined by about 30 % in 1970, and remained at about the same level to 1998. The IS ratio has remained stable over this period. Whereas total DF intake and DF density in Japan are similar to those in Western countries, the IS ratios are higher in Japan. Therefore, the higher incidence of, and mortality from, colon diverticulosis, coronary heart disease, hyperlipidemia, etc., which are all thought to be related to fiber deficiency, in Western countries compared to Japan might be due to the differences in the IS ratio. Conclusions: A decline in total DF intake and DF density is predicted for Japan in the future, because these parameters were lower among the younger generation. This may be due to the marked changes in the dietary habits of the younger generation, and is a problematic trend for Japanese health. Received: 26 April 2002, Accepted: 22 August 2002 Correspondence to: Shigeyuki Nakaji, MD, PhD