BackgroundMatrix metalloproteinases (MMPs) are a family of structural and functional related endopeptidases. They play a crucial role in tumor invasion and building of metastatic formations because of their ability to degrade extracellular matrix proteins. Under physiological conditions their activity is precisely regulated in order to prevent tissue disruption. This physiological balance seems to be disrupted in cancer making tumor cells capable of invading the tissue. In breast cancer different expression levels of several MMPs have been found.MethodsTo fill the gap in our knowledge about MMP expression in breast cancer, we analyzed the expression of all known human MMPs in a panel of twenty-five tissue samples (five normal breast tissues, ten grade 2 (G2) and ten grade 3 (G3) breast cancer tissues). As we found different expression levels for several MMPs in normal breast and breast cancer tissue as well as depending on tumor grade, we additionally analyzed the expression of MMPs in four breast cancer cell lines (MCF-7, MDA-MB-468, BT 20, ZR 75/1) commonly used in research. The results could thus be used as model for further studies on human breast cancer. Expression analysis was performed on mRNA and protein level using semiquantitative RT-PCR, Western blot, immunohistochemistry and immunocytochemistry.ResultsIn summary, we identified several MMPs (MMP-1, -2, -8, -9, -10, -11, -12, -13, -15, -19, -23, -24, -27 and -28) with a stronger expression in breast cancer tissue compared to normal breast tissue. Of those, expression of MMP-8, -10, -12 and -27 is related to tumor grade since it is higher in analyzed G3 compared to G2 tissue samples. In contrast, MMP-7 and MMP-27 mRNA showed a weaker expression in tumor samples compared to healthy tissue. In addition, we demonstrated that the four breast cancer cell lines examined, are constitutively expressing a wide variety of MMPs. Of those, MDA-MB-468 showed the strongest mRNA and protein expression for most of the MMPs analyzed.ConclusionMMP-1, -2, -8, -9, -10, -11, -12, -13, -15, -19, -23, -24, -27 and -28 might thus be associated with breast cancer development and tumor progression. Therefore, these MMPs are proper candidates for further functional analysis of their role in breast cancer.

Trophoblast cell (CTB) invasion into the maternal endometrium plays a crucial role during human embryo implantation and placentation. As for all invasive cell types, the ability of CTB to infiltrate the uterine wall is facilitated by the activity of matrix metalloproteinases (MMPs), which is regulated by tissue inhibitors of MMPs (TIMPs). There is evidence for the expression of several MMPs and TIMPs in decidua. However, published data are limited. Therefore, to set a foundation for future research, we screened a panel of healthy human deciduas obtained during first, second and third trimester of pregnancy in addition to isolated decidual cell populations for the expression of all known human MMPs and TIMPs by RT-PCR, western blot and immunohistochemistry. In the decidual samples, we detected almost all MMPs and all four TIMPs at mRNA level. While the expression of proMMP-3 and active MMP-13 and -23 was down-regulated in the course of pregnancy, the pro forms of MMP-8, -19 and -23, active MMP-9, -10, -12, -15, -16, -26 and -28, and pro- and active MMP-14 increased towards the end of gestation. All MMPs and TIMPs were expressed in uterine natural killer cells, decidual fibroblasts and/or trophoblasts, with the exception of MMP-20 and -25. In summary, a remarkably broad spectrum of MMPs was expressed at the human feto-maternal interface, reflecting the highly invasive and remodelling effect on placenta formation. It can be speculated that expression of MMPs correlates with the invasive potential of CTBs together with a crucial role in activation of labour at term.

Glioblastomas are characterized by an aggressive local growth pattern, a marked degree of invasiveness and poor prognosis. Tumor invasiveness is facilitated by the increased activity of proteolytic enzymes which are involved in destruction of the extracellular matrix of the surrounding healthy brain tissue. Elevated levels of matrix metalloproteinases (MMPs) were found in glioblastoma (GBM) cell-lines, as well as in GBM biopsies as compared with low-grade astrocytoma (LGA) and normal brain samples, indicating a role in malignant progression. A careful review of the available literature revealed that both the expression and role of several of the 23 human MMP proteins is controversely discussed and for some there are no data available at all. We therefore screened a panel of 15 LGA and 15 GBM biopsy samples for those MMPs for which there is either no, very limited or even contradictory data available. Hence, this is the first complete compilation of the expression pattern of all 23 human MMPs in astrocytic tumors. This study will support a better understanding of the specific expression patterns and interaction of proteolytic enzymes in malignant human glioma and may provide additional starting points for targeted patient therapy.

BackgroundMatrix metalloproteinases (MMPs) are involved in the degradation of protein components of the extracellular matrix and thus play an important role in tumor invasion and metastasis. Their expression is related to the progression of gynecological cancers (e.g. endometrial, cervical or ovarian carcinoma). In this study we investigated the expression pattern of the 23 MMPs, currently known in humans, in different gynecological cancer cell lines.MethodsIn total, cell lines from three endometrium carcinomas (Ishikawa, HEC-1-A, AN3 CA), three cervical carcinomas (HeLa, Caski, SiHa), three chorioncarcinomas (JEG, JAR, BeWo), two ovarian cancers (BG-1, OAW-42) and one teratocarcinoma (PA-1) were examined. The expression of MMPs was analyzed by RT-PCR, Western blot and gelatin zymography.ResultsWe demonstrated that the cell lines examined can constitutively express a wide variety of MMPs on mRNA and protein level. While MMP-2, -11, -14 and -24 were widely expressed, no expression was seen for MMP-12, -16, -20, -25, -26, -27 in any of the cell lines. A broad range of 16 MMPs could be found in the PA1 cells and thus this cell line could be used as a positive control for general MMP experiments. While the three cervical cancer cell lines expressed 10-14 different MMPs, the median expression in endometrial and choriocarcinoma cells was 7 different enzymes. The two investigated ovarian cancer cell lines showed a distinctive difference in the number of expressed MMPs (2 vs. 10).ConclusionsIshikawa, Caski, OAW-42 and BeWo cell lines could be the best choice for all future experiments on MMP regulation and their role in endometrial, cervical, ovarian or choriocarcinoma development, whereas the teratocarcinoma cell line PA1 could be used as a positive control for general MMP experiments.

Abstract. NDRG1 is a member of the N-myc downregulated gene (NDRG) family. Its induction occurs via diverse physiological and pathological conditions (hypoxia, cellular differentiation, heavy metal, N-myc, neoplasia) which modulate NDRG1 transcription, mRNA stability and translation. Hypoxia, among other factors, induces NDRG1 expression and plays an important role in its regulation of expression. To date, the complete detailed function of this protein in humans remains unknown. Hypoxia represents a common feature of solid tumors. In our study, differences in NDRG1 expression between different WHO grades of astrocytic tumors were comparatively examined in vivo in human low-grade astrocytoma (WHO grade 2) and glioblastoma (WHO grade 4) at both the protein and mRNA level by Western blot analysis and semi-quantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1· protein and mRNA regulation under hypoxia was also determined in vitro in U251, U373 and GaMG cells. This regulation was shown at the same levels in vivo in human low-grade astrocytoma (WHO grade 2) and glioblastoma which showed a higher NDRG1 overexpression level in glioblastoma than in lowgrade astrocytoma. siRNA-and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements (HREs) bound by nuclear HIF-1· in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. Due to its clear regulatory behavior under hypoxic condition in human tumor cells, NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma.

BackgroundGlioblastomas (GBM), the most frequent malignant brain tumors in adults, are characterized by an aggressive local growth pattern and highly invasive tumor cells. This invasion is facilitated by expression of matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases. They mediate the degradation of protein components of the extracellular matrix. Twenty-three family members are known. Elevated levels of several of them have been reported in GBM. GBM cell-lines are used for in vitro studies of cell migration and invasion. Therefore, it is essential to know their MMP expression patterns. Only limited data for some of the cell-lines are published, yet. To fill the gaps in our knowledge would help to choose suitable model systems for analysis of regulation and function of MMPs during GBM tumorigenesis, cell migration and invasion.FindingsWe analysed MMP-1, -8, -9, -10, -11, -13, -17, -19, -20, -21, -23, -24, -26, -27, and MMP-28 expression in seven GBM cell-lines (SNB-19, GaMG, U251, U87, U373, U343, U138) and in four primary cell cultures by semiquantitative RT-PCR, followed changes in the MMP expression pattern with increasing passages of cell culture and examined the influence of TNF-α and TGF-β1 stimulation on the expression of selected MMPs in U251 and U373 cells.MMP-13, -17, -19 and -24 were expressed by all analyzed cell-lines, whereas MMP-20 and MMP-21 were not expressed by any of them. The other MMPs showed variable expression, which was dependent on passage number. Primary cells displayed a similar MMP-expression pattern as the cell-lines. In U251 and U373 cells expression of MMP-9 and MMP-19 was stimulated by TNF-α. MMP-1 mRNA expression was significantly increased in U373 cells, but not in U251 cells by this cytokine. Whereas TGF-β1 had no impact on MMP expression in U251 cells, it significantly induced MMP-11 and MMP-24 expression in U373 cells.ConclusionsLiterature-data and our own results suggest that the expression pattern of MMPs is highly variable, dependent on the cell-line and the cell-culture conditions used and that also regulation of MMP expression by cytokines is cell-line dependent. This is of high impact for the transfer of cell-culture experiments to clinical implementation.

Hypoxia is a crucial factor in tumour aggressiveness and its treatment resistance, particularly in human brain cancer. Tumour resistance against radiation- and chemo- therapy is facilitated by oxygenation reduction at tumour areas. HIF-1α regulated genes are mostly responsible for this type of resistance. Among these genes, carbonic anhydrase isoform 9 (CA9) is highly overexpressed in many types of cancer especially in high grade brain cancer like GBM. CA IX contributes to tumour environment acidification by catalyzing the carbon dioxide hydration to bicarbonate and protons, leading to the acquisition of metastasic phenotypes and chemoresistance to weakly basic anticancer drugs and therefore to inadequate application of radio-therapeutic or chemotherapeutic anti-cancer treatment strategies. Inhibition of this enzymatic activity by application of specific chemical CA9 inhibitors (sulphonamide derivative compounds) or indirect inhibitors like HIF-1α inhibitors (chetomin) or molecular inhibitors like CA9-siRNA leads to reversion of these processes, leading to the CA9 functional role inhibition during tumourigenesis. Hypoxia significantly influences the tumour microenvironment behaviour via activation of genes involved in the adaptation to the hypoxic stress. It also represents an important cancer prognosis indicator and is associated with aggressive growth, malignant progression, metastasis and poor treatment response. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Sulfonamide derivative compounds with CA9 inhibitory characteristics represent one of the optimal treatment options beside other CA9 inhibitory agents or chemical inhibitory compounds against its main regulating transcription factor which is the hypoxia induced HIF-1α when applied against human cancers with hypoxic regions like GBM, bearing potential for an effective role in human brain tumour therapeutic strategies. Glycolytic inhibitors, when added in controlled doses under hypoxia, lead to a reduced accumulation of HIF-1α and can function as indirect hypoxia regulated genes inhibitors like CA9. These may be used as alternative or in conjunction with other direct inhibitors like the sulphonamide derivate compounds, chetomin or specific siRNAs, or other different chemical compounds possessing similar functionality making them as optimal tools for optimized therapy development in cancer treatment, especially against human brain cancer. Further experimental analysis towards the tumour stage specific inhibitory CA9 characteristics determination are necessary to find the optimal therapeutic solutions among the different available modalities; whether they are direct or indirect chemical, molecular or natural inhibitors to be able to set up successful treatment approaches against the different human tumour diseases.

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