Signal-pathway profiling of cells undergoing photodynamic therapy with reverse phase liquid protein arrays.

3645 Aim: Clinical application of 5-aminolevulinic acid (ALA) results in a tumor specific accumulation of the heme precursor protoporphyrin IX (PPIX). Photodynamic therapy (PDT) using PPIX as the photosensitizer allows treatment of solid tumors as well as early stages by killing cells via singlet oxygen generation. This technique is clinically established. However the mode of cell death leaves many questions unanswered. Apoptosis is favored over necrosis for clinical practice as the latter causes side-effects. Therefore we have analyzed the PDT-induced cell death and its correlation to various PDT parameters in vitro . Due to the high sample number and the sensitivity required, the PDT-induced signal pathways were analyzed with reverse phase liquid protein arrays. Methods: Human cell lines (CaCo2, HT29, UROtsa, RT4, J82) with different degrees of differentiation were cultured in plateau phase and subjected to in vitro- PDT with cell-specific LD 50 and LD 90 doses. Cells were then harvested in a time course by protein lysis. The lysates were arrayed onto nitrocellulose covered glass slides (FAST-slides, Schleicher & Schuell, Germany) by aim of a ring-needle arrayer (417 GMS arrayer, MWG-Biotech). The arrays were analyzed for expression of apoptosis related proteins by immuno histochemistry (antibodies Cell Signaling, MA, USA; DAKO Autostainer, CSA-System Peroxidase-Kit, Dako, Germany). Arrays were digitally scanned and analyzed densitometrically. Results: Analyzing the expression of caspase-3, 7, 9 (each total and cleaved) and AKT, ERK, BAD (each total and phosphorylated) reveals a dose response of signal activation to PDT-stimulus. Higher differentiated phenotypes show a more distinct signal response in general and a higher apoptotic response in detail. Well differentiated CaCo2 showed clearest apoptotic signaling in caspase-3 activation of all cell lines examined whereas moderately well differentiated HT29 died by necrosis (all LD 90 ). Conclusions: Reverse phase liquid protein arrays are a promising technique for signal pathway profiling: They outrange traditional western blots by sensitivity, high throughput capability, minimal sample consumption and easy quantification of results obtained. Data of apoptotic response in higher differentiated cells correlate with recent literature. In addition independent own findings of PDT-response for these particular cell lines could be verified, proofing the protein arrays to be a reliable analytical tool.