Genistein suppresses the invasive potential of human breast cancer cells through transcriptional regulation of metalloproteinases and their tissue inhibitors.

Progression of breast cancer implicates the degradation of extracellular matrix (ECM) by metallo-proteinases (MMPs), a process with important consequences on the growth and invasiveness of cancer cells in adjacent and distant sites. The isoflavone, genistein--a natural inhibitor of protein tyrosine kinase pathway--inhibits the growth of a wide range of cancer cells in vitro. The aim of this study was to investigate: i) the expression of mRNAs encoded for MMPs and their endogenous inhibitors (TIMPs) associated with pathogenesis and metastatic potential of breast cancer cells; and ii) the effect of genistein on the transcription of MMPs and TIMPs and the invasive potential of breast cancer cells. Gene expression at transcriptional level was examined in cell cultures of two epithelial breast cancer cell lines, the high invasive (ER-negative) MDA-MB-231 and the low invasive (ER-positive) MCF-7, as well as the normal mammary cells (MCF-12A) following RNA isolation and reversed transcriptase polymerase chain reaction (RT-PCR). The inhibitory effect of genistein on functional invasiveness was examined by a cell invasion assay. Cell cycle distribution showed that genistein arrested breast cancer MDA-MB-231, MCF-7 and BT-20 cells in the G2/M phase. Both normal and breast cancer cell lines express the genes of MMP-2, -9, MT1-, MT2-, MT3-MMP and TIMP-1, -2 and -3. MCF-7 express notably less MMPs than MDA-MB-231 cell line. The addition of genistein resulted in down-regulation of the transcription of all MMP genes in MDA-MB-231 and most of MMPs in MCF-7 cells. The inhibitory effect of genistein on MMPs was functionally confirmed, since it significantly reduced the invasion properties of cancer cells in vitro. The obtained results indicate that genistein may be of great value in prevention of breast cancer cell metastasis, since it represents both a transcriptional modulator of genes involved in this pathogenetic process and a suppressor of breast cancer cell invasiveness.

STI571 as a potent inhibitor of growth and invasiveness of human epithelial breast cancer cells.

STI571, a specific tyrosine kinase inhibitor, exhibits a substantial therapeutic activity in patients with chronic myeloid leukaemia and gastrointestinal stromal tumors. In this study we examined the activity of STI571 on the growth and invasiveness of three human epithelial breast cancer cell lines of low (MCF-7) and high (ZR-75-1 and MDA-MB-231) invasive potential. Growth of all cell lines in serum-containing medium was significantly inhibited by STI571 in a dose-dependent manner, with an average IC50 of approximately 5-6 microM. Flow cytometric analysis revealed that this effect is characterized by an accumulation of all breast cancer cell types tested in the G2/M-phase of the cell cycle with a concomitant decrease of the percentage of cells in the S-phase. Interestingly, no increase in apoptosis was observed, indicating that the effect of this kinase inhibitor is cytostatic rather than cytotoxic. In addition, STI571 exerts a significant inhibition effect on the invasion of the highly invasive breast cancer cell lines ZR-75-1 and MDA-MB-231. These results encourage further preclinical investigations on the mechanisms underlying the inhibitory effects of STI571, which may be of great value in breast cancer treatment.

Analysis of glycosaminoglycan-derived disaccharides in biologic samples by capillary electrophoresis and protocol for sequencing glycosaminoglycans.

Glycosaminoglycans are biologically significant carbohydrates which either as free chains (hyaluronan) or constituents of proteoglycans (chondroitin/dermatan sulfates, heparin, heparan sulfate and keratan sulfate) participate and regulate several cellular events and (patho)physiological processes. Capillary electrophoresis, due to its high resolving power and sensitivity, has been successfully used for the analysis of glycosaminoglycans. Determination of compositional characteristics, such as disaccharide sulfation pattern, is a useful prerequisite for elucidating the interactions of glycosaminoglycans with matrix effective molecules and, therefore, essential in understanding the biological functions of proteoglycans. The interest in the field of characterization of such biologically important carbohydrates is soaring and advances in this field will signal a new revolution in the area of glycomics equivalent to that of genomics and proteomics. This review focuses on the capillary electrophoresis methods used to determine the disaccharide pattern of glycosaminoglycans in various biologic samples as well as advances in the sequence analysis of glycosaminoglycans using both chromatographic and electrophoretic techniques.

Identification, quantification and fine structural characterization of glycosaminoglycans from uterine leiomyoma and normal myometrium.

The type, amount and fine chemical composition of glycosaminoglycans (GAGs) present both in human normal myometrium and uterine leiomyoma have been studied. GAGs were fractionated by ion-exchange chromatography on DEAE-Sephacel, isolated by gel-permeation chromatography on Sepharose CL-6B and characterized using electrophoresis in cellulose acetate membranes, specific enzymic treatments and analysis by high-performance capillary electrophoresis (HPCE). No statistical intrabatch differences in total GAG content in both tissues were identified, whereas significant interbatch differences between normal myometrium and uterine leiomyoma were recorded. Hyaluronan (HA), chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate (HS) and keratan sulphate (KS) were identified in both tissues. Statistically significant (P

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis.

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.