The characterization of an intestine-like genomic signature maintained during Barrett's-associated adenocarcinogenesis reveals an NR5A2-mediated promotion of cancer cell survival.

Barrett's oesophagus (BO), an intestinal-type metaplasia (IM), typically arising in conjunction with gastro-oesophageal reflux disease, is a prominent risk factor for the development of oesophageal adenocarcinoma (OAC). The molecular similarities between IM and normal intestinal tissues are ill-defined. Consequently, the contribution of intestine-enriched factors expressed within BO to oncogenesis is unclear. Herein, using transcriptomics we define the intestine-enriched genes expressed in meta-profiles of BO and OAC. Interestingly, 77% of the genes differentially expressed in a meta-profile of BO were similarly expressed in intestinal tissues. Furthermore, 85% of this intestine-like signature was maintained upon transition to OAC. Gene networking analysis of transcription factors within this signature revealed a network centred upon NR5A2, GATA6 and FOXA2, whose over-expression was determined in a cohort of BO and OAC patients. Simulated acid reflux was observed to induce the expression of both NR5A2 and GATA6. Using siRNA-mediated silencing and an NR5A2 antagonist we demonstrate that NR5A2-mediated cancer cell survival is facilitated through augmentation of GATA6 and anti-apoptotic factor BCL-XL levels. Abrogation of NR5A2-GATA6 expression in conjunction with BCL-XL co-silencing resulted in synergistically increased sensitivity to chemotherapeutics and photo-dynamic therapeutics. These findings characterize the intestine-like signature associated with IM which may have important consequences to adenocarcinogenesis.

Drug Discovery Approaches Utilizing Three-Dimensional Cell Culture.

Historically, two-dimensional (2D) cell culture has been the preferred method of producing disease models in vitro. Recently, there has been a move away from 2D culture in favor of generating three-dimensional (3D) multicellular structures, which are thought to be more representative of the in vivo environment. This transition has brought with it an influx of technologies capable of producing these structures in various ways. However, it is becoming evident that many of these technologies do not perform well in automated in vitro drug discovery units. We believe that this is a result of their incompatibility with high-throughput screening (HTS). In this study, we review a number of technologies, which are currently available for producing in vitro 3D disease models. We assess their amenability with high-content screening and HTS and highlight our own work in attempting to address many of the practical problems that are hampering the successful deployment of 3D cell systems in mainstream research.

Lead Structures for Applications in Photodynamic Therapy. 6. Temoporfin Anti-Inflammatory Conjugates to Target the Tumor Microenvironment for In Vitro PDT.

Due to the ongoing development of clinical photodynamic therapy (PDT), the search continues for optimized photosensitizers that can overcome some of the side effects associated with this type of treatment modality. The main protagonists being: post-treatment photosensitivity, due to only limited cellular selectivity and post-treatment tumor regrowth, due to the up-regulation of pro-inflammatory agents within the tumor microenvironment. A photosensitizer that could overcome one or both of these drawbacks would be highly attractive to those engaged in clinical PDT. Certain non-steroidal anti-inflammatory drugs (NSAIDs) when used in combination with PDT have shown to increase the cytotoxicity of the treatment modality by targeting the tumor microenvironment. Temoporfin (m-THPC), the gold standard chlorin-based photosensitizer (PS) since its discovery in the 1980's, has successfully been conjugated to non-steroidal anti-inflammatory compounds, in an attempt to address the issue of post-treatment tumor regrowth. Using a modified Steglich esterification reaction, a library of "iPorphyrins" was successfully synthesized and evaluated for their PDT efficacy.

Lead structures for applications in photodynamic therapy. 5. Synthesis and biological evaluation of water soluble phosphorus(V) 5,10,15,20-tetraalkylporphyrins for PDT.

Improved photosensitizers for use in photomedicine must possess good water-solubility and optimal photophysical properties. Phosphorus(V) porphyrins fulfill these criteria and are a class of porphyrins with significant potential applications in phototherapy. Five phosphorus(V) porphyrins bearing alkyl substituents have been synthesized. Reasonable to good yields were obtained for all P(V) insertions and all compounds underwent biological evaluation for their PDT activity on two esophageal cancer cell lines, OE33 and SKGT-4. Their cellular uptake was investigated using a high content screening method. Notably, three compounds displayed good uptake and using the MTS cell proliferation assay, two were shown to have photocytotoxicity comparable to mTHPC (Temoporfin(®)) with IC50 values of 6.5 and 5.5 µM.

High content screening as high quality assay for biological evaluation of photosensitizers in vitro.

A novel single step assay approach to screen a library of photdynamic therapy (PDT) compounds was developed. Utilizing high content analysis (HCA) technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4). The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT). The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells). A high correlation was found between the high content screening (HCS) and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR) study for photosensitizer libraries.

Transferrin conjugation does not increase the efficiency of liposomal Foscan during in vitro photodynamic therapy of oesophageal cancer.

Photodynamic therapy (PDT) is based on the delivery of photocytotoxic agents to a target tissue, followed by irradiation. In order to increase the efficiency of PDT in oesophageal cancer therapy, polyethylene glycol (PEG)-grafted, transferrin (Tf)-conjugated liposome formulations of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (Foscan), a second-generation photosensitiser, were prepared. Expression of transferrin receptors (CD71) in the oesophageal cancer cell line, OE21, was confirmed by immunoblot and confocal laser scanning microscopy. The anti-proliferative effect of Foscan liposomes was evaluated and compared with plain formulations (i.e., without Tf) as well as with free drug. In addition, the intracellular accumulation was studied using high content analysis. Surprisingly, delivering Foscan by transferrin-conjugated PEG-liposomes to oesophageal cancer cells did not improve the photocytotoxicity or the intracellular accumulation of Foscan when compared to unmodified liposomes or indeed free photosensitiser. Tf-targeted drugs and drug delivery systems have shown improved the therapy of many cancers. Our study, however, did not corroborate these findings. If this is due to the tumour type, the choice of in vitro model or the delivery systems remains to be confirmed.

Synthesis and biological evaluation of a library of glycoporphyrin compounds.

A library of glycosylated porphyrins (glycoporphyrins) was prepared and the compounds were evaluated for their photodynamic therapy (PDT) activity against the oesophageal squamous-cell carcinoma cell line OE21 in vitro. A synthetic methodology was developed to allow incorporation of biologically active carbohydrates, including the histo-blood-group antigen trisaccharide Lewis(X), onto the porphyrin backbone. The effect of the carbohydrate group and substitution pattern on the PDT activity, cell uptake and subcellular localisation of the glycoporphyrin compounds is reported.

Synthesis and evaluation of the europium(III) and zinc(II) complexes as luminescent bioprobes in high content cell-imaging analysis.

Novel phenanthroline derivatives and their europium(III) and zinc(II) complexes have been prepared in up to 92%. In contrast to the stable zinc complexes, the europium compounds exhibit a strong luminescence in THF solution. However, quenching of the emission is observed in DMSO indicating complete dissociation of the complexes back to free ligands in this solvent. (1)H NMR studies of the Eu(III)-complexes 5 and 6 also confirmed the existence of different states depending on the solvent used. Moreover, it was found that compound 5 is stable in EtOH-PBS solutions; here a strong signal in the emission spectra corresponding to the europium ion was detected. No spectral changes were observed for the zinc(II) complexes, they were shown to be stable in the media. These metal complexes can be used as fluorescence markers for the diagnosis of oesophageal squamous carcinoma (OE21) cells at low concentrations. Cell images were acquired using the compounds 5, 7-9 as luminescent agents. The first images were taken already after 20 min incubation time at a very low concentration range (0.7-1.6 µM).

Simple but powerful: phenanthroline-based small molecules for cellular imaging and cancer screening.

A two-step synthetic procedure gives highly fluorescent phenanthroline molecular probes. The compounds localize in the endoplasmic reticulum and their potential as bioactive probes was evaluated. The materials are quickly taken up by living cells within 5 min. Preliminary in vitro studies have shown that these compounds are selective to esophageal cancer cells and can be used as selective markers in intracellular cancer diagnostics. The materials show a remarkable cytotoxicity towards cancer cells vs normal as 7-1.