Cell-type Specific Protoporphyrin IX Metabolism in Human Bladder Cancer in vitro¶

Abstract

5-Aminolevulinic acid (ALA)-supported fluorescence endoscopy of the urinary bladder results in a detection rate of bladder cancer superior to that of white light endoscopy. The different accumulation of the metabolite protoporphyrin IX (PPIX) in tumor cells after ALA instillation is poorly understood; however, it is crucial to optimize diagnosis and potential phototherapy. For systematic analysis of cell-type specific PPIX accumulation and metabolism two human bladder carcinoma cell lines (RT4 and J82), a normal urothelial cell line (UROtsa), and a fibroblast cell line (N1) were chosen, and grown in two different growth states to model important tissue components of the urinary bladder, i.e. tumor, normal epithelium and stroma. To quantitate PPIX content, fluorescence intensities measured by flow cytometry were matched with cellular PPIX extraction values, and related to relative ferrochelatase activity, cellular iron content, number of transferrin receptors per cell and porphobilinogen deaminase (PBGD) activity. For in vitro experiments, the initial correlation of relative flow cytometric and spectrometric measurements of PPIX provides a calibration curve for consequent flow cytometric PPIX quantification. Lower fluorescence of normal cells could be explained by significant differences of ferrochelatase activity and iron content in comparison to tumor cells. However, the content of iron was not related to transferrin receptor content. PBGD activity seemed to play a minor role for the differential accumulation of PPIX in urothelial cells. In conclusion, the in vitro culture of urothelial cells and fibroblasts indicates that the most important metabolic step for PPIX accumulation in the urinary bladder is the transition from PPIX to heme. Further investigation of PPIX metabolism does support the validation of photodynamic diagnosis, and might also lead the way to a highly specific tumor related molecule.

Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

“…ALA uptake, heme synthesis, and intra-cellular traffic of porphyrins. The definitive mechanisms of these factors were defined by detailed biochemical analysis [6]–[12]. However, the mechanism of PpIX efflux from mitochondria to the cytoplasm has not been clarified despite the fact that PpIX is synthesized inside mitochondria.…”

“…Despite the importance of PpIX in ALA-mediated PDD and PDT, the mechanism of accumulation of this metabolite in cancer cells remains unclear. The intracellular localization and concentrations of protoporphyrins are determined by at least three rate-limiting steps, namely ALA uptake through proton-coupled oligopeptide transporters, such as PEPT1 and PEPT2 [8]–[10], conversion of PpIX to heme (ferrochelatase, FECH) [11], [12], and intracellular traffic of PpIX through ATP-binding cassette (ABC) transporter G2 (ABCG2) [13], [14]. ABCG2, also called breast cancer resistant protein, was first discovered in doxorubicin-resistant breast cancer cells [15], and it predominantly localizes to the plasma membrane [16].…”

Chemically triggered release of 5-aminolevulinic acid from liposomes

“…Addition of exogenous 5-ALA leads to selective accumulation of PpIX in cells undergoing high metabolic turnover, including cancer cells. 2, 3 5-ALA is finding increasing clinical use as an adjunct in fluorescence-guided surgery, where it is especially effective for guiding resection of high grade glioma. 4 Low intensity, blue light illumination of the surgical field produces a two-color fluorescence image that helps surgeons delineate tumor margins.…”

ALA- and ALA-hexylester-induced protoporphyrin IX fluorescence and distribution in multicell tumour spheroids

“…They demonstrated that PpIX is synthesized less effectively under hypoxia than in normoxic conditions, with the magnitude of the effect of hypoxia being strongly dependent on cell density and proliferation status. The complexity of the relationships among various factors influencing PpIX accumulation is reflected in a recent report of Krieg et al (2000), which showed that changes in PpIX content observed when normal urothelial, fibroblast and 2 human bladder cancer cell lines were cultured in subconfluent vs postconfluent conditions could be interpreted on the basis of intracellular iron content and ferrochelatase activity. In a welldifferentiated tumour cell line, ferrochelatase activity increased significantly in postconfluent culture conditions, while in a poorly differentiated line, the ferrochelatase activity decreased when cell culture conditions were changed from subconfluent (exponential) to postconfluent (plateau).…”