Abstract
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.
Highlights
High content screening (HCS) is a powerful tool for the biological evaluation of potentially therapeutic compounds and widely used in drug discovery, biomedical research and pharmaceutical industry
A final screen of 22 new compounds dissolved in dimethyl sulfoxide (DMSO) was performed in OE21, SKGT-4 and OE33 cell lines using a single concentration of 2 mM, for this OE33 and SKGT-4 cells were plated at a concentration of 66103 cells per well and OE21 cells were used in the previously disclosed concentration
In order to overcome these apparent limitations here we present the results obtained for a novel high content assay developed using fluorescent drugs in OE21 cells, a human esophageal squamous cell carcinoma cell line derived from a human carcinoma
Summary
High content screening (HCS) is a powerful tool for the biological evaluation of potentially therapeutic compounds and widely used in drug discovery, biomedical research and pharmaceutical industry This high throughput technique is based on high resolution microscopy and multi-parametric automated image analysis allowing a rapid quantitative evaluation of drug candidates on a large scale [1,2]. One of its branches includes photodynamic therapy (PDT), which is successfully used to treat different medical conditions including cancer for over 40 years [8,9,10,11] It is based on the accumulation of a photosensitizing drug (PS) in the target tissue, which generates toxic singlet oxygen and other reactive oxygen species upon irradiation with light [7]. Most experimental manipulations with the living cellular materials have to be carried out under special illumination conditions [7,16,17]. 16 By their very nature these compounds have an intrinsic fluorescence over a broad range of excitation and emission wavelengths making difficult to use conventional assays that are broadly used for a drug evaluation
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