Previous studies have shown that low-dose PDT using 5-aminolevulinic acid (ALA)-induced photoporphyrin IX
(PpIX) can induce apoptosis in tumor cells without causing necrosis. In this study we investigated the molecular
mechanisms associated with apoptosis after ALA-PDT treatment in two brain glioma cell lines: human U87, and rat
CNS-1cells. We used high energy light at a short time (acute PDT) and low energy light at a long time of exposure
(metronomic PDT) to treat both cell lines. The cells were treated with 0.25 mM ALA at 5 joules for energy. We
found that CNS-1 cells were more resistant to ALA-PDT than U87 cells when treated by both acute and metronomic
PDT. To screen possible apoptosis mechanisms associated with acute and metronomic PDT, microarray analysis of
gene expression was performed on RNA from glioblastoma cells treated with either acute or metronomic ALA-PDT.
Within the set of genes that were negatively or positively regulated by both treatments are tumor necrosis
factor receptors. The expression of TNF receptors was investigated further by RT-PCR and western blotting. The
apoptosis mechanism of the cell death occurred through different pathways including BCL-2 and TNF receptors,
and in part caused by cleaving caspase 3. Interestingly, metronomic ALA-PDT inhibited the expression of LTβR
and the transcription factor NFκB. This inhibition was ALA concentration dependent at low concentrations.
This paper reports further studies of Radiation Induced Fibrosarcoma (RIF-1) tumor cells which have been made resistant to Photodynamic Therapy by multiple treatment and regrowth in vitro using the hematoporphyrin derivative photosensitizer Photofrin. Previous work has shown both structural and functional changes in the mitochondria of the resistant (RIF-8A) cells. Colocalization of Photofrin and the mitochondrial localizer Rhodamine-123 was assessed by double-label confocal fluorescence microscopy (CFM). At 18h Photofrin incubation, there was strong correlation in discrete subcellular sites between Photofrin and Rhodamine fluorescence. However, in RIF-8A cells there were also discrete regions of Rhodamine localization which showed weak or no Photofrin fluorescence. This was not observed in RIF-1 cells. CFM measurements also showed that the Photofrin fluorescence after 18h incubation was reduced by increasing concentration of Rhodamine (30 min. incubation), and that this dependence was different for the two cell types. The RIF-8A cells were also shown to be cross-resistant to cisplatin and to have an associated reduced level of Pt-DNA adducts, suggesting the possibility of increased repair capacity. Cross-resistance was not observed, however, with a ruthenium phthalocyanine photosensitizer nor, as previously reported, with other chemotherapeutic agents such as Adriamcyin. Thus, there is a complex pattern of cross-resistance with these cells. Preliminary observations of the effects of a respiratory chain inhibitor (oligomycin) and an uncoupler of oxidative phosphorylation (FCCP) indicate differences between RIF-8A and RIF-1 which may be related to the condensed mitochondrial structure of the RIF-8A cells.