Identification of benzo(a)pyrene diol epoxide-binding DNA fragments using DNA immunoprecipitation technique

Benzo(a)pyrene diol epoxide (BPDE), an active metabolite of the tobacco carcinogen benzo(a)pyrene, can induce p53 gene mutation, down-regulate retinoic acid receptor beta, and increase cyclooxygenase-2 expression in human epithelial cells. However, it remains unknown whether these effects are direct or indirect. To investigate the direct effects of BPDE on gene expression, we used our newly developed DNA immunoprecipitation technique to identify and clone BPDE-binding DNA fragments. A total of 67 fragments were sequenced and grouped into four categories after their sequences were blasted in the GenBank database: (a). 15 fragments matched known gene sequences; (b). 24 matched expressed sequence tag clones; (c). 22 matched genomic DNA of unknown genes; and (d). 6 clones did not show any homology with GenBank sequences known to date. The 67 fragments include DNA repair and apoptosis-related genes, zinc finger protein, cellular enzymes, expressed sequence tag clones, and CpG islands. These data further demonstrate that BPDE-induced gene alterations are important events in carcinogenesis and that the identification of the resulting clones may help us to better understand the mechanisms of BPDE-induced carcinogenesis.     

Methylation of the thyroid-stimulating hormone receptor gene in epithelial thyroid tumors: a marker of malignancy and a cause of gene silencing

Thyroid-stimulating hormone receptor (TSHR) expression is frequently silenced in epithelial thyroid cancers associated with decreased or absent TSH-promoted iodine uptake. To study the underlying molecular mechanism of decreased TSHR expression, we examined the methylation status of the TSHR gene promoter by sequencing bisulfite-treated DNA from thyroid tumors. After identification of methylated sites by sequencing bisulfite-treated DNA, we used methylation-specific polymerase chain reaction and found frequent CpG methylation in papillary thyroid cancer (23 of 39 patients; 59%) and follicular thyroid cancers (7 of 15 patients; 47%). In contrast, we saw no methylation in normal thyroid tissues and benign adenomas (0 of 8 patients; 0%). In human thyroid tumor cell lines, we observed that TSHR was normally expressed at the protein and mRNA level in cells where the TSHR gene was unmethylated, whereas it was silenced in cell lines where the TSHR promoter was hypermethylated. Treatment of the latter cells with a demethylating agent partially restored TSHR expression. We thus demonstrate aberrant methylation of human TSHR as a likely molecular pathway responsible for the silencing of this gene in thyroid cancers. We propose that methylation of TSHR may provide a novel diagnostic marker of malignancy and a basis for potential use of demethylating agents in conjunction with TSH-promoted radioiodine therapy for epithelial thyroid cancers.     

Antitumor effect by interleukin-11 receptor alpha-locus chemokine/CCL27, introduced into tumor cells through a recombinant adenovirus vector

In this study, we examined antitumor activity of a mouse CC chemokine ILC/CCL27 and a mouse CX(3)C chemokine fractalkine/CX(3)CL1 in vivo. We generated recombinant adenovirus vectors with a fiber mutation, encoding mILC (Ad-RGD-mILC) and mFKN (Ad-RGD-mFKN). We confirmed tumor cells infected with Ad-RGD-mILC and Ad-RGD-mFKN to express and release these chemokines. Tumor rejection experiments in vivo were carried out by inoculating OV-HM cells infected with Ad-RGD-mILC or Ad-RGD-mFKN into immunocompetent mice. mILC significantly suppressed the tumor growth, whereas no such significant effect was observed by mFKN. The antitumor activity induced by mILC was T cell dependent, involving both CD4(+) and CD8(+) T cells. Immunohistochemical analysis revealed accumulation of both CD3(+) lymphocytes and NK cells in the tumor tissue transduced with mILC and mFKN. However, there was a significant difference in the distribution of infiltrating cells. Furthermore, mFKN appeared to have an angiogenic activity, which might have masked its tumor suppressive activity. Collectively, ILC/CCL27 may be a good candidate molecule for cancer gene therapy.     

Involvement of the p38 mitogen-activated protein kinase cascade in hepatocellular carcinoma

BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is activated in response to various extracellular stimuli. The authors investigated the involvement of the p38 MAPK, a member of the MAPK superfamily, cascade in hepatoma cell lines and in human hepatocellular carcinoma (HCC) tissue specimens. METHODS: Constitutively active mutant of MAPK kinase 6 (MKK6), which is upstream of p38 MAPK, was transfected into the HepG2 and HuH7 human hepatoma cell lines. The constitutive active mutant was constructed by replacing Ser-189 and Thr-193 with Glu. The growth and death of mutant MKK6-transfected hepatoma cells were analyzed by the WST-1 and sub-G1 assays. The surgically resected livers of 20 HCC patients were divided histologically into tumorous (T) and nontumorous (NT) lesions. p38 MAPK activity was analyzed using in vitro kinase assay and MKK6 activity was measured using Western blot analysis. RESULTS: Mutant MKK6 transfection increased p38 MAPK activity, cytochrome c release from the mitochondria to the cytosol, and caspase-3 activity, accompanied by apoptosis. In contrast, SB203580, a p38 MAPK-specific inhibitor, prevented MKK6-induced apoptosis in hepatoma cell lines. In the T lesions of 20 HCC parients, p38 MAPK and MKK6 activities were significantly lower compared with NT lesions (P < 0.05). There was a significant positive correlation between p38 MAPK and MKK6 activity (r = 0.507, P < 0.05). Larger tumors (> 20 mm) exhibited lower levels of p38 MAPK and MKK6 activity than did smaller tumors (P < 0.05). CONCLUSIONS: These findings suggested that reduction of the p38 MAPK cascade may account, in part, for the resistance to apoptosis, leading to the unrestricted cell growth of human HCC.     

Bulbinelonesides A-E,phenylanthraquinone glycosides from the roots of Bulbinella floribunda

The roots of Bulbinella floribunda have been analyzed for the phenolic constituents, resulting in the isolation of five new phenylanthraquinone glycosides, named bulbinelonesides A-E (1-5), along with two known phenylanthraquinones, (+)-M-knipholone (6) and (+)-M-isoknipholone (7). The structures of the new compounds were determined on the basis of extensive spectroscopic analysis, including 2D NMR, and the results of enzymatic hydrolysis. Although the new compounds 3-5, whose absolute stereochemistry of the unsymmetric biaryl moiety was determined to be P by the CD spectrum, did not show apparent cytotoxicity against cultured HSC-2 tumor cells and HPC normal cells, the new compounds 1 and 2, as well as the known compounds 6 and 7, whose biaryl bond was assigned as M, exhibited a tumor-specific cytotoxicity against HSC-2 cells comparable to or slightly weaker than etoposide, used as a positive control.     

Eranthisaponins A and B,two new bisdesmosidic triterpene saponins from the tubers of Eranthis cilicica

The present investigation aimed at the glycoside constituents of the tubers of Eranthis cilicica has resulted in the isolation of two new bisdesmosidic triterpene saponins based upon hederagenin, named eranthisaponins A (1) and B (2), along with four known triterpene saponins. The structures of the new saponins 1 and 2 were determined on the basis of spectroscopic analysis, including extensive 1D and 2D NMR data, and acid hydrolysis followed by chromatographic analysis. This is the first report concerning the secondary metabolites of E. cilicica.     

Inhibitory effects of triterpenes isolated from Hoelen on free radical-induced lysis of red blood cells

Hoelen, sclederma of Poria cocos Wolf, has long been used as a sedative and diuretic in traditional medicine. Formerly, we demonstrated that Hoelen in vitro protects red blood cells from AAPH-induced hemolysis. In this study, tests were carried out to identify the main ingredient of Hoelen that has the scavenging effect on free-radicals. Triterpene carboxylic acids isolated from the methanol extract of Hoelen, i.e. pachymic acid, polyporenic acid, 3-epidehydrotumulosic acid, 3beta-hydroxylanosta-7,9(11), 24-trien-21-oic acid and 3-o-acetyl-16 alpha -hydroxytrametenolic acid, were found to have inhibitory activities against AAPH-induced lysis of red blood cells.     

Glypican-3, overexpressed in hepatocellular carcinoma,modulates FGF2 and BMP-7 signaling

The Glypican (GPC) family is a prototypical member of the cell-surface heparan sulfate proteoglycans (HSPGs). The HSPGs have been demonstrated to interact with growth factors, act as coreceptors and modulate growth factor activity. Here we show that based on oligonucleotide array analysis, GPC3 was upregulated in hepatocellular carcinoma (HCC). By northern blot analysis, GPC3 mRNA was found to be upregulated in 29 of 52 cases of HCC (55.7%). By Western blot analysis carried out with a monoclonal anti-GPC3 antibody we generated, the GPC3 protein was found to be overexpressed in 6 hepatoma cell lines, HepG2, Hep3B, HT17, HuH6, HuH7 and PLC/PRF/5, as well as 22 tumors (42.3%). To investigate the role of overexpressed GPC3 in liver cancer, we analyzed its effects on cell growth of hepatoblastoma-derived cells. Overexpression of GPC3 modulated cell proliferation by inhibiting fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 7 (BMP-7) activity. An interaction of GPC3 and FGF2 was revealed by co-immunoprecipitation, while GPC3 was found to inhibit BMP-7 signaling through the Smad pathway by reporter gene assay. The modulation of growth factors by GPC3 may help explain its role in liver carcinogenesis. In addition, the ability of HCC cells to express GPC3 at high levels may serve as a new tumor marker for HCC.