Recent evidence shows that transcriptional silencing as a consequence of hypermethylation of CpG islands is an important mechanism in the inactivation of p16INK4 tumor suppressor gene. This study is designed to clarify the significance of p16INK4 hypermethylation in 23 cases of glioblastomas (GBMs) by methylation-specific polymerase chain reaction (PCR) and p16 immunostaining. Fourteen cases (60.9%) out of 23 GBMs revealed hypermethylation on p16. p16 immunostaining revealed that 13 (93%) of these 14 hypermethylation cases showed complete loss of immunoreactivity and only one (7%) case retained immunoreactivity. Among 9 methylation-negative cases, 4 were immunonegative, which might be related to mutations or deletions other than hypermethylation. The most significant finding was that of 17 cases with immunonegativity, 13 cases (76.5%) showed hypermethylation. We reconfirmed that p16 hypermethylation may be one of the major mechanisms of tumorigenesis of GBMs and the results between the methylation specific-PCR study and p16 immunostaining had a good correlation.
5' Untranslated Regions/metabolism* ; 5' Untranslated Regions/genetics ; Adult ; Antisense Elements (Genetics) ; Brain Neoplasms/pathology ; Brain Neoplasms/genetics* ; Brain Neoplasms/chemistry ; CpG Islands/physiology* ; DNA Methylation* ; Female ; Gene Silencing/physiology ; Glioblastoma/pathology ; Glioblastoma/genetics* ; Glioblastoma/chemistry ; Human ; Male ; Middle Age ; Polymerase Chain Reaction ; Protein p16/genetics* ; Protein p16/analysis

AIM: To study the chemical constituents of the rhizomes of Alpinia officinarum Hance. METHOD: Compounds were isolated by repeated column chromatography, and their structures were elucidated on the basis of spectral analysis. The cytotoxic activities of these compounds were evaluated with the T98G and B16F10 cell lines by the MTT assay. RESULTS: A dimeric diarylheptanoid, named alpinin B (1), along with three known diarylheptanoids were obtained, and their structures were identified as alpinin B (1), 1, 7-diphenyl-3,5-heptanedione (2), (4E)-1, 7-diphenylhept-4-en-3-one (3) and (4E)-7- (4-hydroxyphenyl)-1-phenylhept-4-en-3-one (4). CONCLUSION Compound 1 is a new dimeric diarylheptanoid. The biosynthetic pathway of 1 was speculated to originate from a Michael reaction between compounds 2 and 3. Compound 3 showed cytotoxicity against the human glioblastoma T98G cell line with IC50 of 27 μmol·L(-1).
Alpinia ; chemistry ; Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; therapeutic use ; Cell Line, Tumor ; Diarylheptanoids ; chemistry ; isolation & purification ; pharmacology ; therapeutic use ; Glioblastoma ; drug therapy ; Humans ; Molecular Structure ; Phytotherapy ; Plant Extracts ; chemistry ; pharmacology ; therapeutic use ; Rhizome ; chemistry

OBJECTIVETo study the mechanism of antisense epidermal growth factor receptor cDNA in growth suppression of glioblastomas cells.

METHODSGlioblastoma U87MG cells, which over-express epidermal growth factor receptor (EGFR), were transfected with antisense-EGFR constructs. Several clones with stable expression of lower or undetectable levels of EGFR protein were obtained. The effect of antisense-EGFR on cell differentiation was studied using morphological evaluation and western blotting analysis of glial fibrillary acidic protein (GFAP) expression. The effect of antisense-EGFR on cell cycle was studied by flow cytometry and immunohistochemical analysis of p53, Rb, p16 and CDK4 expressions. The effect of antisense-EGFR on telomerase activity was studied by telomeric repeat amplification protocol (TRAP) assay.

RESULTSU87MG cells that were transfected with antisense-EGFR constructs had smaller cell bodies and longer processes, and expressed higher level of GFAP compared with that of the control cells. Flow cytometric analysis showed that the proportion of cells in G(0)/G(1) phases of the cell cycle in the antisense EGFR cDNA transfected clones increased significantly when compared with control cells, whereas the proportion of cells in S phase decreased markedly. In addition, immunohistochemical analysis showed that the expression of wild-type p53 was significantly increased in the antisense-EGFR cDNA transfected clones, whereas the expressions of Rb, p16 and CDK4 were not altered. TRAP assay revealed that telomerase activity in the antisense-EGFR clones was significantly decreased.

CONCLUSIONSAntisense-EGFR transfection inhibits U87MG cell growth by inducing cell differentiation and p53 expression, G(1) cell cycle arrest and inhibition of telomerase activity.

Cell Line, Tumor ; DNA, Antisense ; therapeutic use ; DNA, Complementary ; therapeutic use ; Flow Cytometry ; Glioblastoma ; chemistry ; drug therapy ; pathology ; Humans ; Immunohistochemistry ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; genetics ; Retinoblastoma Protein ; analysis ; Transfection ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVETo investigate the correlation of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U87 as well as the effect of EGFR-cDNA transfection on the expression of bcl-2 and Bax in U87 cells.

METHODSThe correlation of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U87 was studied by confocal microscopy and Western blot. The expression of bcl-2 and Bax in EGFR-cDNA transfected and untransfected glioblastoma cell lines was studied by Western blot.

RESULTSConfocal microscopic images showed that bcl-2 protein was localized in the periphery of the nuclear matrix and Bax in the nuclear matrix. A 26 kDa bcl-2 band and a specific band of Bax at about 66 000 were detected in nuclear matrix proteins by western blot. The expression of bcl-2 was lower but that of Bax was higher in EGFR-cDNA transfected cells than the control.

CONCLUSIONBcl-2 and Bax, being nuclear matrix associated proteins, are probably involved in the EGFR-cDNA induced malignant conversion of glioblastoma cells by introducing EGFR cDNA into the tumor cells.

Apoptosis ; Cell Line, Tumor ; Glioblastoma ; pathology ; Humans ; Nuclear Matrix ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; physiology ; Receptor, Epidermal Growth Factor ; genetics ; physiology ; Transfection ; bcl-2-Associated X Protein ; analysis ; physiology