BACKGROUNDHyaluronidase (Hyase) is an enzyme which hydrolyses hyaluronan (HA), a large nonsulfated glycosaminoglycan. Several genes have been identified to code for hyaluronidases in humans. Its role has only recently been underlined in the invasion of prostate cancer, colonic cancer, and breast cancer. Moreover, the findings were in agreement with some experimental results which showed that HA-derived oligosaccharides had angiogenesis-promoting activity. All these findings prompted us to investigate factors that had been characterized as putative invasive factors in different human breast cancer-derived cell lines.

METHODSWe selected two series of human breast cancer-derived cell lines whose expression of estrogen receptors (ER) was previously published. Hyaluronidase secretion in culture medium and expression of matrix metallo-proteinase (MMP)-9, cathepsin-D (cath-D) and vascular endothelial growth factor (VEGF) by cells were determined. We also investigated cell invasiveness in the Matrigel invasion assay, and studied the capability of cancer cells to promote in vitro formation of tubules by endothelial cells.

RESULTSER(-) cells secreted significantly more hyaluronidase (P < 0.001) and expressed significantly more VEGF (P < 0.01), MMP-9 (P < 0.05) and cath-D (P < 0.0001) than ER(+) cells. Invasion through Matrigel by ER(-) Hyase(+) cells was significantly higher than that by ER(+) Hyase(-) cells (P < 0.05). In both cases, invasion was decreased by heparin (P < 0.05). When ECV-304 endothelial cells were co-cultivated in millicell chambers with cancer cells, ECV-304 cells were induced to form tubules. Tubule formation was demonstrated to be more prominent with ER(-) Hyase(+) cells than with ER(+) Hyase(-) cells (P < 0.05).

CONCLUSIONInvasive features of ER(-) breast cancer cells can be characterized in vitro by an invasive Matrigel assay, as the induction of tubule formation by ECV-304 endothelial cells, higher secretion of hyaluronidase, and higher expression of proteinases MMP-9, cath-D, and the angiogenesis promoting factor VEGF.

Breast Neoplasms ; metabolism ; Cathepsin D ; metabolism ; Cell Line, Tumor ; Humans ; Hyaluronoglucosaminidase ; metabolism ; Immunohistochemistry ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; genetics ; Receptors, Estrogen ; genetics ; Vascular Endothelial Growth Factor A ; metabolism

The presence of hyl gene in 364 PFGE clones of Enterococcus faecium was detected by colony hybridization under conditions of high stringency. The isogenic hyl-deficient mutant (* hyl) was constructed with suicide pTX4577 and screened by allelic replacement. Moreover, an in vitro study was made on the effect of hyl gene detection on the growth ability of hylgene detection on the mutant, and an in vivo study was made on the decrease of virulence in the mouse peritonitis model. The results showed, in the clinical isolates, the positive percentage of hyl gene was 32.8%, which was significantly higher than that (5.3%) in the non-clinical isolates. The * hyl was selected by kanamycin and identified by PCR, pulsed field gel electrophoresis (PFGE) and Southern blot. The experimental evidence indicated that the growth ability of * hyl was remarkably reduced in comparison with that of the wild-type strain. The percentage survival of mice in TX2466 groups was 0, while that of * hyl groups was 50% at the same inoculum in mouse peritonitis. The differences were significant. These data suggest that hyl gene in specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Being important in the pathogenesis, hyl gene is probably a major virulence factor of Enterococcus faecium.
Animals ; Enterococcus faecium ; genetics ; pathogenicity ; Genes, Bacterial ; Gram-Positive Bacterial Infections ; microbiology ; Hyaluronoglucosaminidase ; genetics ; Male ; Mice ; Mice, Inbred ICR ; Mutation ; Peritonitis ; microbiology ; Virulence ; Virulence Factors ; genetics ; metabolism

OBJECTIVETo evaluate the expression pattern of PH20 in primary and metastatic breast cancer and its relationship to tumor metastatic potential.

METHODSAnti-PH20 antibody was synthesized by injection of conjugated human PH20 peptides into rabbits. Immunohistochemical study was performed on 53 cases of human breast cancer. Western blot was used to detect PH20 expression in 5 cases of breast cancer with available fresh tissue. Two oligonucleotide probes were prepared for in-situ hybridization using breast tissue microarray.

RESULTSNormal breast tissue did not express PH20 (0/3), while 58.4% (31/53) of breast cancer cases did. The highest expression rate was found in metastatic foci in regional lymph nodes (83.3%), followed by primary breast cancer tissue in cases with lymph node secondaries (70.8%). The breast cancer cases with no any metastasis had an expression rate of 48.2%. The immunohistochemical staining results were further confirmed by Western blotting. In-situ hybridization showed PH20 RNA in 75% of the breast cancer tissue (21/28). Two of the 17 cases of normal breast tissue showed weak expression in some ductolobular units.

CONCLUSIONSThe expression of PH20 has a positive correlation with metastatic potential in breast cancer. It is possible that PH20 may play an important role in the invasive growth and metastasis of breast cancer cells, via mechanisms such as digestion of surrounding stromal tissue and release of FGF-2.

Adenocarcinoma, Mucinous ; metabolism ; pathology ; Adult ; Animals ; Breast ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Female ; Humans ; Hyaluronoglucosaminidase ; biosynthesis ; genetics ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; biosynthesis ; genetics ; Rabbits

OBJECTIVETo investigate the inhibitory effect of hyaluronic acid (HA), hyaluronidase (Hase) and arg-gly-asp tripeptide (RGD) on adhesion and invasion of human gastric cancer cell line SGC7901.

METHODSExpression of CD44 and integrin beta1 protein on cell surface was determined by indirect fluorescence. After SGC7901 cells were treated with HA, Hase and RGD alone or in various combinations, their adhesion and invasion to ECM were measured by MTT and Boyden chamber method. Cell morphology was also observed.

RESULTSExpression of CD44 and integrin beta1 protein on cell surface was detected in human gastric cancer cell line SGC7901. Hase or RGD alone could block the adhesion and invasion of SGC7901 cells to ECM in contrast to the control (P < 0.001, P < 0.05); their blocking effect was stronger than HA (P < 0.05). The inhibitory effect of Hase + HA or a combination of the three agents was stronger than any single agent (P < 0.001). Morphologically, the untreated cells adhered onto the matrigel had spread out presenting a fibroblast feature with variously shaped pseudopods, while the treated ones were kept round with relatively fewer pseudopods.

CONCLUSIONHA, Hase and RGD can inhibit the adhesion and invasion of SGC7901 cells expressing functional CD44 and integrin beta1 protein to ECM, and a combination of the three agents may achieve the best inhibitory effect.

Adenocarcinoma ; metabolism ; pathology ; Animals ; Antineoplastic Agents ; pharmacology ; Cell Adhesion ; drug effects ; Cell Line, Tumor ; Drug Synergism ; Humans ; Hyaluronan Receptors ; metabolism ; Hyaluronic Acid ; pharmacology ; Hyaluronoglucosaminidase ; pharmacology ; Integrin beta1 ; metabolism ; Mice ; NIH 3T3 Cells ; Neoplasm Invasiveness ; Oligopeptides ; pharmacology ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVE: To assess the value of DNA methylation level of HYAL2 gene as a molecular marker for differential diagnosis of malignant and benign thyroid tumors. METHODS: DNA methylation of HYAL2 gene in tissue specimens of 190 patients with papillary thyroid cancer (PTC) and 190 age- and gender-matched patients with benign thyroid tumors was examined by mass spectrometry, and the protein expression of HYAL2 was detected immunohistochemically for another 55 pairs of patients. Logistic regression analysis was performed to calculate the odds ratio (OR) and evaluate the correlation of per 10% reduction in DNA methylation with PTC. Receiver operating characteristic (ROC) curve analysis was performed and the area under curve (AUC) was calculated to assess the predictive value of alterations in HYAL2 methylation. RESULTS: Hypomethylation of HYAL2_CpG_3 was significantly correlated with early-stage PTC (OR=1.51, P=0.001), even in stage I cancer (OR=1.42, P=0.007). Age-stratified analysis revealed a significantly stronger correlation between increased HYAL2_CpG_ 3 methylation and early-stage PTC in patients below 50 years than in those older than 50 years (OR: 1.89 vs 1.37, P < 0.05); ROC analysis also showed a larger AUC of 0.787 in younger patients. The results of immunohistochemistry showed that patients with PTC had significantly higher protein expressions of HYAL2 than patients with benign tumors. CONCLUSION The alterations of DNA methylation level of HYAL2 gene is significantly correlated with early-stage PTC, suggesting the value of DNA methylation level as a potential biomarker for differentiation of malignant from benign thyroid tumors.
Adenoma, Oxyphilic/genetics* ; Biomarkers, Tumor/metabolism* ; Cell Adhesion Molecules/metabolism* ; DNA Methylation ; GPI-Linked Proteins/metabolism* ; Humans ; Hyaluronoglucosaminidase/metabolism* ; Immunohistochemistry ; Middle Aged ; Thyroid Cancer, Papillary/pathology* ; Thyroid Neoplasms/pathology*

OBJECTIVETo study the effect of SiRNA-EGFR on the expression of hyaluronidase gene in human breast cancer cells.

METHODSReverse transcription-polymerse chain reaction was used to detect the changes in the expression of EGFR mRNA in human breast cancer cell lines MDA-MB-231, MDA-MB-435S, ZR-75 and ZR-75-30 after transfection by SiRNA-EGFR.

RESULTSAfter transfection with SiRNA-EGFR, the expression levels of EGFR were significantly inhibited in MDA-MB-231, MDA-MB-435S, ZR-75 and ZR-75-30 cells (P<0.05).

CONCLUSIONTransfection by SiRNA-EGFR can inhibit the expression of EGFR mRNA in human breast cancer cells.

Breast Neoplasms ; enzymology ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Humans ; Hyaluronoglucosaminidase ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Transfection

Acid Anhydride Hydrolases ; metabolism ; Apoptosis ; Biomarkers, Tumor ; metabolism ; Caspases ; metabolism ; Cell Adhesion Molecules ; metabolism ; Chromosome Deletion ; Drug Delivery Systems ; GPI-Linked Proteins ; metabolism ; Humans ; Hyaluronoglucosaminidase ; metabolism ; In Situ Hybridization, Fluorescence ; methods ; Lung Neoplasms ; diagnosis ; drug therapy ; genetics ; metabolism ; Neoplasm Proteins ; metabolism ; Neoplasm Staging ; Receptor, Epidermal Growth Factor ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

Various kinds of glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known to be involved in structural and space-filling functions, as well as many physiological regulations in skin. To investigate ultraviolet (UV) radiation-mediated regulation of GAGs and PGs in cultured human dermal fibroblasts, transcriptional changes of many types of PGs and GAG chain-synthesizing enzymes at 18 hr after 75 mJ/cm2 of UV irradiation were examined using quantitative real-time polymerase chain reaction methods. Hyaluronic acid synthase (HAS)-1, -2, and -3 and hyaluronidase-2 mRNA expressions were significantly increased by UV irradiation. Expressions of lumican, fibromodulin, osteoglycin, syndecan-2, perlecan, agrin, versican, decorin, and biglycan were significantly decreased by UV irradiation, while syndecan-1 was increased. Expressions of GAG chain-synthesizing glycosyltransferases, xylosyltransferase-1, beta1,3-glucuronyltransferase-1, beta1,4-galactosyltransferase-2, -4, exostosin-1, chondroitin polymerizing factor, and chondroitin sulfate synthase-3 were significantly reduced, whereas those of beta1,3-galactosyltransferase-6, beta1,4-galactosyltransferase-3, -7, beta-1,3-N-acetylglucosaminyltran sferase-2, and -7 were increased by UV irradiation. Heparanase-1 mRNA expression was increased, but that of heparanase-2 was reduced by UV irradiation. Time-course investigation of representative genes showed consistent results. In conclusion, UV irradiation may increase hyaluronic acid production through HAS induction, and decrease other GAG productions through downregulation of PG core proteins and GAG chain-synthesizing glycosyltransferases in cultured human dermal fibroblasts.
Cell Line ; Fibroblasts/metabolism/radiation effects ; Gene Expression Regulation/radiation effects ; Glucuronosyltransferase/genetics/radiation effects ; Glycosaminoglycans/*biosynthesis/chemistry ; Glycosyltransferases/genetics/*metabolism ; Humans ; Hyaluronic Acid/biosynthesis ; Hyaluronoglucosaminidase/genetics/radiation effects ; Polymerase Chain Reaction ; Proteoglycans/*biosynthesis/genetics/radiation effects ; RNA, Messenger/analysis/genetics ; Skin/*metabolism/radiation effects ; Transcription, Genetic/radiation effects ; *Ultraviolet Rays