Sandra Liekens

The dual role of thymidine phosphorylase in cancer development and chemotherapy.

Thymidine phosphorylase (TP), also known as “platelet‐derived endothelial cell growth factor” (PD‐ECGF), is an enzyme, which is upregulated in a wide variety of solid tumors including breast and colorectal cancers. TP promotes tumor growth and metastasis by preventing apoptosis and inducing angiogenesis. Elevated levels of TP are associated with tumor aggressiveness and poor prognosis. Therefore, TP inhibitors are synthesized in an attempt to prevent tumor angiogenesis and metastasis. TP is also indispensable for the activation of the extensively used 5‐fluorouracil prodrug capecitabine, which is clinically used for the treatment of colon and breast cancer. Clinical trials that combine capecitabine with TP‐inducing therapies (such as taxanes or radiotherapy) suggest that increasing TP expression is an adequate strategy to enhance the antitumoral efficacy of capecitabine. Thus, TP plays a dual role in cancer development and therapy: on the one hand, TP inhibitors can abrogate the tumorigenic and metastatic properties of TP; on the other, TP activity is necessary for the activation of several chemotherapeutic drugs. This duality illustrates the complexity of the role of TP in tumor progression and in the clinical response to fluoropyrimidine‐based chemotherapy.

CXCL12-CXCR4 Axis in Angiogenesis, Metastasis and Stem Cell Mobilization

Chemokines are key players in the attraction and activation of leukocytes and are thus implicated in the recruitment of immune cells at sites of infection and/or inflammation. They exert their action by binding to seven-transmembrane G protein-coupled receptors. The chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 represents the single natural ligand for the chemokine receptor CXCR4. CXCL12 possesses angiogenic properties and is involved in the outgrowth and metastasis of CXCR4-expressing tumors and in certain inflammatory autoimmune disorders, such as rheumatoid arthritis. CXCR4 expression on tumor cells is upregulated by hypoxia and angiogenic factors, such as vascular endothelial growth factor (VEGF). CXCR4 also acts as a co-receptor for entry of human immunodeficiency virus (HIV) in CD4(+) T cells. Finally, CXCL12/CXCR4 interactions were shown to play an important role in the migration of hematopoietic stem cells and their progenitors from, and their retention within, the bone marrow, a site characterized by high CXCL12 expression. As such, CXCR4 inhibitors may be utilized to inhibit HIV-1 infection, tumor growth and metastasis and to mobilize hematopoietic stem cells from the bone marrow in the circulation, where they can be collected for autologous stem cell transplantation. Here, we discuss the different aspects of CXCL12/CXCR4 biology as well as the development and anti-cancer/stem cell mobilizing activity of CXCR4 antagonists.

CXCR3 ligands in disease and therapy

Chemokines, binding their various G protein-coupled receptors, lead the way for leukocytes in health and inflammation. Yet chemokine receptor expression is not limited to leukocytes. Accordingly, chemokines are remarkably pleiotropic molecules involved in a range of physiological as well as pathological processes. For example, the CXCR3 chemokine receptor is expressed on activated T lymphocytes, dendritic cells and natural killer cells, but also fibroblasts and smooth muscle, epithelial and endothelial cells. In men, these cells express either CXCR3A, its splice variant CXCR3B or a balanced combination of both. The CXCR3 ligands, activating both receptor variants, include CXCL4, CXCL4L1, CXCL9, CXCL10 and CXCL11. Upon CXCR3A activation these ELR-negative CXC chemokines mediate chemotactic and proliferative responses, for example in leukocytes. In contrast, CXCR3B induces anti-proliferative and anti-migratory effects, as exemplified by angiostatic effects on endothelial cells. Taken together, the unusual and versatile characteristics of CXCR3 and its ligands form the basis for their pertinent involvement in a myriad of diseases. In this review, we discuss the presence and function of all CXCR3 ligands in various malignant, angiogenic, infectious, inflammatory and other disorders. By extension, we have also elaborated on the potential therapeutic applicability of CXCR3 ligand administration or blockade, as well as their additional value as predictive or prognostic biomarkers. This review illustrates the multifunctional, intriguing character of the various CXCR3-binding chemokines.

Novel pyrazole derivatives: Synthesis and evaluation of anti-angiogenic activity

The synthesis of a series of novel trisubstituted pyrazole derivatives and their PIFA-mediated conversion to molecules bearing the fused pyrazolo[4,3-c]quinoline ring system is reported. The anti-angiogenic activity of these compounds was evaluated by using in vitro assays for endothelial cell proliferation and migration, and in the chicken chorioallantoic membrane (CAM) assay. Compounds containing the fused pyrazolo[4,3-c]quinoline motifs emerged as potent anti-angiogenic compounds, which also had the ability to inhibit the growth of human breast (MCF-7) and cervical (Hela) carcinoma cells in vitro.

Phosphoramidate ProTides of the Anticancer Agent FUDR Successfully Deliver the Preformed Bioactive Monophosphate in Cells and Confer Advantage over the Parent Nucleoside

The fluorinated pyrimidine family of nucleosides continues to represent major current chemotherapeutic agents for treating solid tumors. We herein report their phosphate prodrugs, ProTides, as promising new derivatives, which partially bypass the dependence of the current drugs on active transport and nucleoside kinase-mediated activation. They are also resistant to metabolic deactivation by phosphorolytic enzymes. We report 39 ProTides of the fluorinated pyrimidine FUDR with variation in the aryl, ester, and amino acid regions. Notably, only certain ProTide motifs are successful in delivering the nucleoside monophosphate into intact cells. We also find that the ProTides retain activity in mycoplasma infected cells, unlike FUDR. Data suggest these compounds to be worthy of further progression.

Zebrafish Bioassay-Guided Natural Product Discovery: Isolation of Angiogenesis Inhibitors from East African Medicinal Plants

Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications

In this paper we report on the controlled biofunctionalization of the hydrophobic layer of electrowetting-on-dielectric (EWOD) based microfluidic chips with the aim to execute (adherent) cell-based assays. The biofunctionalization technique involves a dry lift-off method with an easy to remove Parylene-C mask and allows the creation of spatially controlled micropatches of biomolecules in the Teflon-AF(®) layer of the chip. Compared to conventional methods, this method (i) is fully biocompatible; and (ii) leaves the hydrophobicity of the chip surface unaffected by the fabrication process, which is a crucial feature for digital microfluidic chips. In addition, full control of the geometry and the dimensions of the micropatches is achieved, allowing cells to be arrayed as cell clusters or as single cells on the digital microfluidic chip surface. The dry Parylene-C lift-off technique proves to have great potential for precise biofunctionalization of digital microfluidic chips, and can enhance their use for heterogeneous bio-assays that are of interest in various biomedical applications.

Antitumor Potential of Acyclic Nucleoside Phosphonates

Acyclic nucleoside phosphonates such as HPMPC (cidofovir) and PMEA (adefovir) have been identified as broad-spectrum antiviral agents that are effective against herpes-, retro- and hepadnavirus infections (PMEA) and herpes-, pox-, adeno-, polyoma-, and papillomavirus infections (HPMPC). Here we show that HPMPC and PMEA also offer great potential as antitumor agents, through the induction of tumor cell differentiation (PMEA), inhibition of angiogenesis (HPMPC) and induction of apoptosis (HPMPC). In vivo tumor regressions have been noted for choriocarcinoma (PMEA) in rats, hemangioma (HPMPC) in rats and papillomatous lesions (HPMPC) in humans. Acyclic nucleoside phosphonates can be considered as a new dimension to the discipline of chemotherapy. They have a unique mode of action that is targeted at (viral or tumoral) DNA synthesis. They exhibit a pronounced and prolonged anti-viral and/or tumoral activity that can persist for days or weeks after a single administration. Most importantly, they have a uniquely broad spectrum of indications for clinical use, encompassing both DNA- and retrovirus infections, as well as various forms of cancer of both viral and non-viral origin.

Inhibition of angiogenesis by blockers of volume-regulated anion channels

Osmotic cell swelling activates an outwardly rectifying Cl(-) current in endothelial cells that is mediated by volume-regulated anion channels (VRACs). In the past, we have shown that serum-induced proliferation of endothelial cells is arrested in the presence of compounds that potently block the endothelial VRACs. Here we report on the effects of four chemically distinct VRAC blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid] (NPPB), mibefradil, tamoxifen, and clomiphene-on several models of experimental angiogenesis. Mibefradil (20 microM), NPPB (100 microM), tamoxifen (20 microM), and clomiphene (20 microM) inhibited tube formation by rat microvascular endothelial cells plated on matrigel by 42.9 +/- 8.8%, 25.3 +/- 10.4%, 32.2 +/- 4.5%, and 20 +/- 5.8%, respectively (p < 0.05). Additionally, NPPB (50-100 microM) and mibefradil (10-30 microM) significantly inhibited bFGF (10 ng/ml) + TNFalpha (2.5 ng/ml)-stimulated microvessel formation by human microvascular endothelial cells plated on fibrin by 30-70%. Furthermore, NPPB, mibefradil, and clomiphene concentration dependently inhibited spontaneous microvessel formation in the rat aorta-ring assay and vessel development in the chick chorioallantoic membrane assay. These results suggest that VRAC blockers are potent inhibitors of angiogenesis and thus might serve as therapeutic tools in tumor growth and other angiogenesis-dependent diseases.

7-Deazaxanthine, a novel prototype inhibitor of thymidine phosphorylase

7‐Deazaxanthine (7DX) was identified as a novel inhibitor of thymidine (dThd) phosphorylase (TPase). It inhibited the TPase reaction in a concentration‐dependent manner. At 1 mM, it almost completely prevented the TPase‐catalysed hydrolysis of dThd to thymine. The 50% inhibitory concentration (IC50) of 7DX was 40 μM in the presence of 100 μM of the natural substrate dThd. 7DX is also endowed with a marked inhibitory effect on angiogenesis. It significantly prevents neovascularisation in the chicken chorioallantoic membrane during development. 7DX is the first purine derivative shown to be a potent inhibitor of purified TPase and angiogenesis.