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Nonsteroidal antiinflammatory drugs (NSAIDs) appear to reduce the risk of developing cancer. One mechanism through which NSAIDs act to reduce carcinogenesis is to inhibit the activity of cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in various cancer tissues. Overexpression of COX-2 increases cell proliferation and inhibits apoptosis. However, selective COX-2 inhibitors can also act through COX-independent mechanisms. In this review, we describe the COX-2-independent molecular targets of these COX-2 inhibitors and discuss how these targets may be involved in the anticarcinogenic activities of these selective COX-2 inhibitors. We also compare the concentrations of these inhibitors used in in vitro and in vivo experiments and discuss the implications of the in vitro studies for clinical management of cancer with these drugs.

Beside their structural role for the cell membrane the family of sphingolipids act as effector molecules in signal transduction with links to various aspects of cancer initiation, progression and treatment response. The "sphingolipid rheostat" balances between apoptosis inducing ceramid and growth promoting sphingosine-1-phosphate. We analyzed gene expression of 43 proteins from this pathway in different subtypes of breast cancer using microarray data of 1,269 tumor samples (test set n=171; validation sets n=1098) and observed significant differences for several genes. Sphingosine kinase 1 (SPHK1), ceramide galactosyltransferase (UGT8), and Ganglioside GD3-Synthase (ST8SIA1) displayed higher expression among ER negative tumors. In contrast, glucosylceramidsynthase (GCS), dihydroceramidsynthases (LASS4, LASS 6) and acid ceramidase (ASAH1) were higher expressed in ER positive samples. Survival analysis revealed a worse outcome of patients with high SPHK1 expression. To avoid a confounding effect of the ER status we also restricted the analysis to 750 patients with ER positive tumors. Again a worse outcome was observed for tumors displaying high SPHK1 expression. While 75.8+/-1.9% of the patients with tumors low in SPHK1 expression were free of metastasis at 5 years, this was the case for only 64.9+/-3.6% of patients with tumors displaying high SPHK1 expression (P=0.008). Immunohistochemistry identified the carcinoma cells as the major source of SPHK1 expression in the tumor. The correlation of SPHK1 with a poor prognosis as well as its high expression among ER negative tumors are in line with the antiapoptotic and proliferative properties of its product sphingosine-1-phosphate. Targeting of the sphingolipid rheostat may thus open new treatment options.

Direct quantification of biomolecular interaction by single-molecule force spectroscopy has evolved into a powerful tool for materials and life sciences. We introduce an approach in which the unbinding forces required to break intermolecular bonds are measured in a differential format by comparison with a known reference bond (here, a short DNA duplex). In addition to a marked increase in sensitivity and force resolution, which enabled us to resolve single-base pair mismatches, this concept allows for highly specific parallel assays. This option was exploited to overcome cross-reactions of antibodies in a protein biochip application.

Several in vitro studies have correlated dysfunction of the sphingolipid-signaling pathway with promotion of tumor cell growth as well as progression and resistance of tumors to chemotherapeutic agents. As ceramides (Cer) constitute the structural backbones of all sphingolipids, we investigated the endogenous ceramide levels in 43 malignant breast tumors and 21 benign breast biopsies and compared them with those of normal tissues using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The total ceramide levels in malignant tumor tissue samples were statistically significantly elevated when compared with normal tissue samples. Upregulation of the total ceramide level averaged 12-fold and 4-fold higher than normal tissue samples, for malignant tumors and benign tissues, respectively. Specifically, the levels of C(16:0)-Cer, C(24:1)-Cer and C(24:0)-Cer were significantly raised in malignant tumors as compared with benign and normal tissue. The augmentation of the various ceramides could be assigned to an increase of the messenger RNA levels of ceramide synthases (CerS) LASS2 (longevity assurance), LASS4 and LASS6. Notably, elevated levels of C(16:0)-Cer were associated with a positive lymph node status, indicating a metastatic potential for this ceramide. Moreover, the levels of C(18:0)-Cer and C(20:0)-Cer were significantly higher in estrogen receptor (ER) positive tumor tissues as compared with ER negative tumor tissues. In conclusion, progression in breast cancer is associated with increased ceramide levels due to an upregulation of specific LASS genes.

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Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-Å resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.Riboflavin (vitamin B 2 ) is biosynthesized in plants and numerous microorganisms but not in animals, which depend on nutritional sources. Its derivatives, flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD), are indispensable in all cells where they serve a variety of redox reactions (1). They have also been shown to serve a variety of other functions in cells such as DNA photorepair (2), light sensing (3, 4), and bioluminescence (5-8) and in reactions without net-redox change (9).Gram-negative bacteria are absolutely dependent on endogenous synthesis of riboflavin, because they are devoid of an uptake system for flavins or flavocoenzymes. Hence, riboflavindeficient mutants e.g. of Escherichia coli and Salmonella sp. require extremely high concentrations of exogenous riboflavin for growth. The same is true for yeasts such as Saccharomyces cerevisiae and Candida guilliermondii (for review see Refs. 1 and 10 -13). Thus, these organisms should be vulnerable to inhibitors of the riboflavin biosynthesis, which could therefore qualify as novel anti-infective agents. The absence of the riboflavin pathway in the human host appears advantageous in this context, because host/parasite selectivity of inhibitory agents would not constitute a problem. The mechanistic and structural analysis of the riboflavin pathway could serve as the basis for the rational design of riboflavin pathway inhibitors.3,4-Dihydroxy-2-butanone 4-phosphate synthase supplies the building blocks for the assembly of the xylene ring of the vitamin (14 -17). In fact, all eight carbon atoms of the xylene moiety are derived from the product of the enzyme. In the biosynthetic pathway, 3,4-dihydroxy-2-butanone 4-phosphate is condensed with 5-amino-6-ribitylamino-2,4(1H,3H)-...

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