Gene Expression Pattern following Hypericin-PDT

Photodynamic Tumor Therapy (PDT) is a most promising novel approach to tumor treatment. It is based on the preferential uptake and retention of a photosensitizer by tumors and the production of reactive oxygen species (ROS) during irradiation of the accumulated photosensitizer in the tumor area with visible light. PDT is characterized by both, high tumor specificity and low side effects in contrast to radiation- or chemotherapy. The extract hypericin from Hypericum perforatum plants, commonly known as St. Johns wort, was introduced quite recently as a very powerful photosensitizer; it is successfully being applied in tumor diagnosis and tested for clinical treatment. Up to now, it has not been shown, whether low doses of hypericin may induce apoptosis in tumors to such a great extent as was found in vitro; yet the cell death mode will be crucial for any immunological anti-tumor-response following PDT. However, a deeper knowledge of the steps in the signaling pathways following PDT is still lacking. The understanding of where these intracellular signals originate and how they are integrated and interpreted by the cell will lead to more insight into the proteins that are involved in regulating and executing the cell death program(s) and in modulating immunological anti-tumor mechanisms. The aim of the present project is therefore to uncover the basic processes leading to successful hypericin-PDT by a large scale gene expression analysis following treatment of the squamous cell carcinoma cell line A-431, which will be used as a human tumor cell model. Differentially expressed genes shall be identified on high density cDNA arrays with approximately 10 000 ESTs and a selected set shall be verified by real-time PCR. Knowledge of the expression pattern of cancer cells following PDT with hypericin may lead to novel approaches for enhancing the PDT effects of this promising photosensitizer. In addition, gene expression patterns of hypericin- and ALA-treated A-431 cells (from the preceding project) shall be compared. The differences of progression from photodynamic damage to apoptotic and/or necrotic cell death shall be analyzed and discussed.

Photodynamic tumour therapy (PDT) is a relatively new modality for the treatment of tumorous and non- tumourous lesions as well as for antibacterial and antiviral purposes. It is based on the preferential uptake and retention of a photosensitizer by target cells and the production of reactive oxygen species (ROS) during irradiation of the accumulated photosensitizer in the target cells with visible light of the appropriate wavelength leading the destruction of target cells via apoptosis and/or necrosis. Hypericin, which is extracted from St. John`s wort (Hypericum perforatum) is gaining rapidly in importance as photosensitizer for therapeutical use, since it is very effective in low doses, as well as fluorescence marker for diagnosis due to its bright fluorescence and high selectivity. The aim of the present project was to enlighten the basic molecular biological response of the model system A-431 cells to photodynamic treatment with hypericin (HYP-PDT) in a detailed and comprehensive manner. To meet this directive, gene expression patterns of HYP-PDT treated samples, which were taken at 1.5, 3, 5 and 8 hours post irradiation, as well as appropriate controls were studied using high-density cDNA arrays containing 9738 EST sequences. Verification of array results of selected genes was achieved by real-time PCR analysis on Biorad iCycler system. Clustering of gene expression changes detected on arrays, which were higher than twofold, to functional classes revealed (number of genes upregulated / number of genes downregulated): apoptosis (55/2), oxidative stress (25/4), cytoskeleton and cell attachment (24/12), proliferation and cell cycle (17/9), MAPK signalling pathway (17/5), protein transport (10/2), energy metabolism (8/7), G-protein pathways (6/2), RNA processing (6/2). Real-time PCR verification confirmed 25 genes as upregulated and 8 as downregulated out of 46 selected genes. Strongest downregulation (5.8 fold) showed thrombospondin 1 which is involved in angiogenesis, cell attachment and apoptosis. All other confirmed downregulated genes confirmed only weak expression changes (2-3fold) and are also related to cell attachment. Highest upregulation (>100fold) showed the early stress response genes CL-100, c-Fos, c-Jun and Not. All other confirmed upregulated genes showed only minor changes (most of them <10fold). This comprehensive study of changes in gene expression following HYP-PDT reveals the nature of cellular response to photodynamic treatment of A431 cells with hypericin involving several different molecular pathways (oxidative stress, MAPK, endoplasmatic reticulum stress response) as well as cell attachment, which is in contrast to the results of our previous FWF-funded project dealing with gene expression changes following ALA-PDT (P15143), where, due to the different localization of the sensitizer, mainly necrotic cell death and only very few changes in gene expression patterns were observed.