Clinical and experimental evidence of Bcl-2 involvement in the response to photodynamic therapy.

PURPOSE: The role of apoptosis related proteins in the response of human malignancies to photodynamic therapy (PDT) is under investigation. The aim of the study was to examine the role of p53 and of bcl-2 protein expression in the response to PDT. MATERIALS AND METHODS: Paraffin-embedded material from 37 patients with early esophageal cancer treated with PDT (argon dye laser after intravenous injection of hematoporphyrine derivative) was studied immunohistochemically for p53 protein nuclear accumulation and bcl-2 cytoplasmic expression. Patients with residual disease after two rounds of PDT received definitive radiotherapy. In a subsequent in vitro study, W138 human lung fibroblasts and W138-SV-40 virus transformed were assessed for their sensitivity to PDT. The constitutive bcl-2 overexpression of the transformed cells vs. normal cells (assessed with RT-PCR) was 16-fold. RESULTS: Positive bcl-2 and p53 expression was noted in 10 out of 36 (27%) and 14 out of 36 (39%) patients, respectively. Seven out of 11 tumors (63%) with bcl-2 expression responded completely to PDT vs. 6 out of 26 (23%) of cases with no bcl-2 expression (p = 0.02). No association of p53, T-stage and of histology grade with response to PDT or PDT/RT was noted. The sensitivity to PDT of transformed human fibroblasts compared to normal ones was 4 times more at a fluence of 4.3 J/cm2 (4% vs. 1% cell kill) as well as at a fluence of 5.4 J/cm2 (8% vs. 2% cell kill). CONCLUSION: Bcl-2 protein expression is associated with favorable response to PDT and can be used as a predictor of cancer response to PDT. This finding can be explained by experimental studies showing that PDT induces selective degradation of the bcl-2 protein, leading to apoptosis by decreasing the bcl-2/bax ratio. Studies on PDT combination with agents targeting bcl-2 (i.e. taxanes) are on going to eventually assess a super-additive effect.

Expression kinetics of the (proto) oncogenes c-myc and bcl-2 following photodynamic treatment of normal and transformed human fibroblasts with 5-aminolaevulinic acid-stimulated endogenous protoporphyrin IX.

In the same way as common tumour therapies can cause secondary tumour induction, photodynamic tumour therapy also shows a moderate mutagenicity. The oncogenes responsible for it can be distinguished from their proto-oncogenic precursors by an irreversible increase in their constitutive expression. Transient changes of the expression level of (proto) oncogenes can indicate the beginning of disturbances in the cell homeostasis: many of these genes have a normal function in proliferation or play a role in apoptosis. In this study, therefore, quantitative determination of the expression of the (proto) oncogenes c-myc and bcl-2 in normal and transformed human fibroblasts at different times following photodynamic treatment with 5-aminolaevulinic acid-stimulated endogenous protoporphyrin IX and low-dose irradiation has been carried out by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The aim is to investigate if irreversibly increased (proto) oncogene expression can be found, and if expression changes are involved in cell-cycle alterations (detected in a parallel study) and in initiation of apoptotic processes. The results show: (1) no mutagenic risk, since the over-expression of c-myc and bcl-2 is transient; (2) an interaction of bcl-2 and c-myc associated with an increase of the proliferative activity of the cell cycle of transformed cells; (3) a possible role of bcl-2 in counteracting processes that could be at least precursors for apoptosis induction; and (4) higher constitutive expression of both genes in transformed than in normal fibroblasts.

Analysis of altered gene expression profiles in retinoic acid or CpG-treated Sprague-Dawley rats with MNU-induced mammary adenocarcinoma by cDNA macro array.

In the present work the effects of 13-cis retinoic acid (RA) and CpG-containing oligodeoxynucleotides (CpG-ODN) on the gene expression profile of spleen and tumor tissue in a MNU-induced mammary gland carcinoma ratmodel were investigated by the use of a commercial cDNA macro array (Atlas rat toxicology array 1.2, Clontech). Treatment with these components, either alone or in combination, induced differences of the expression profiles between the distinct treatment groups in both tissues. The large number of genes with altered expression (> 200) points to a highly complex process in vivo.

Molecular response to hypericin-induced photodamage.

Hypericin (Hyp) is used as a powerful natural photosensitizer in photodynamic therapy (PDT). After selective accumulation in tumor tissue, vessels and matrix, and activated by visible light, it destroys the tumor mainly via generation of reactive oxygen species. After photoactivation, molecular biological mechanisms lead to different cellular endpoints: "biostimulation" (increased proliferation rate), repair of the damage leading to rescue of the cells, autophagy, apoptosis and necrosis. Growth stimulation after low-dose Hyp-PDT seems to be induced via the p38 or JNK survival pathways. Since both pathways are also activated by stress, modification of these pathways may also contribute to rescue mechanisms as well as to damage processing. By increasing PDT doses beyond sublethal damage, stress response pathways are activated such as the ER-stress pathway with disruption of Ca2+ homeostasis and unfolded protein response. This leads either to apoptosis or autophagic cell death, dependent on the availability of Bax/Bak. Apoptosis triggered directly at the mitochondria or by the ER-stress response is executed via the mitochondrial pathway, whereas in some cases, the receptor-mediated pathway is preferred. If the damage is too severe, the cellular energy level low and /or the cytoplasma membrane leaky, cells will die necrotically. The different modes of cellular responses depend mainly on the PDT-protocol, photosensitizer localisation, cellular damage protection and the available intracellular energy.