Rene C. Krieg

Metabolic Characterization of Tumor Cell–specific Protoporphyrin IX Accumulation After Exposure to 5-Aminolevulinic Acid in Human Colonic Cells¶

5‐Aminolevulinic acid (ALA)–induced protoporphyrin IX (PPIX) fluorescence has been shown to have high tumor cell selectivity in various organs, including the gastrointestinal (GI) tract. To better understand and to possibly find new approaches to therapeutic application, we investigated the uptake kinetics and consequent metabolism of ALA and PPIX, respectively. Three colon carcinoma (CaCo2, HT29, SW480) and a stromal cell line (fibroblast, CCD18) were chosen to mimic important aspects of malignant mucosa of the GI tract. Because differential PPIX concentrations in these cell lines represented the in vivo observations (ratio tumor vs normal 10:1–20:1), we analyzed the ALA uptake, mitochondrial properties and key molecules of PPIX metabolism (porphobilinogen deaminase [PBGD], ferrochelatase [FC], iron content, transferrin receptor content). The tumor‐preferential PPIX accumulation is strongly influenced, but not solely determined, by activity differences between the PPIX‐producing PBGD and the PPIX‐converting FC, when compared with fibroblasts. Tumor‐specific PPIX accumulation is generated by ALA conversion rather than by initial ALA uptake because no significant overall difference in uptake (about 0.6 μg ALA/mg protein) of ALA is seen. In conclusion, further research of tumor cell selectivity of PPIX fluorescence should focus on the mechanisms responsible for an altered PPIX metabolism to find tumor‐specific target molecules, thus leading to an improved clinical practicability of ALA application and consequent endoscopy.

Clinical Proteomics for Cancer Biomarker Discovery and Therapeutic Targeting

As we emerge into the post-genome era, proteomics finds itself as the driving force field as we translate the nucleic acid information archive into understanding how the cell actually works and how disease processes operate. Even so, the traditionally held view of proteomics as simply cataloging and developing lists of the cellular protein repertoire of a cell are now changing, especially in the sub-discipline of clinical proteomics. The most relevant information archive to clinical applications and drug development involves the elucidation of the information flow of the cell; the “software” of protein pathway networks and circuitry. The deranged circuitry of the cell as the drug target itself as well as the effect of the drug on not just the target, but also the entire network, is what we now are striving towards. Clinical proteomics, as a new and most exciting sub-discipline of proteomics, involves the bench-to-bedside clinical application of proteomic tools. Unlike the genome, there are potentially thousands of proteomes: each cell type has its own unique proteome. Moreover, each cell type can alter its proteome depending on the unique tissue microenvironment in which it resides, giving rise to multiple permutations of a single proteome. Since there is no polymerase chain reaction equivalent to proteomics- identifying and discovering the “wiring diagram” of a human diseased cell in a biopsy specimen remains a daunting challenge. New micro-proteomic technologies are being and still need to be developed to drill down into the proteomes of clinically relevant material. Cancer, as a model disease, provides a fertile environment to study the application of proteomics at the bedside. The promise of clinical proteomics and the new technologies that are developed is that we will detect cancer earlier through discovery of biomarkers, we will discover the next generation of targets and imaging biomarkers, and we can then apply this knowledge to patient-tailored therapy.

Intracellular Localization is a Cofactor for the Phototoxicity of Protoporphyrin IX in the Gastrointestinal Tract: In Vitro Study¶†

Abstract Photodynamic therapy (PDT) is a new treatment modality for solid tumors as well as for flat lesions of the gastrointestinal tract. Although the use of 5-aminolevulinic acid–induced protoporphyrin IX (PPIX) shows important advantages over other photosensitizers, the main mechanisms of phototoxicity induced are still poorly understood. Three human colon carcinoma cell lines with variable degrees of differentiation and a normal colon fibroblast cell line were used to generate a suitable in vitro model for investigation of photosensitizer concentration as well as the applied light dose. Also, the effects of intracellular photosensitizer localization on efficiency of PDT were examined, and cellular parameters after PDT (morphology, mitochondrial transmembrane potential, membrane integrity and DNA fragmentation) were analyzed to distinguish between PDT-induced apoptosis from necrosis. The fibroblast cell line was less affected by phototoxicity than the tumor cells to a variable degree. Well-differentiated tumor cells showed higher toxicity than less-differentiated cells. After irradiation, cell lines with cytosolic or mitochondrial PPIX localization indicate a loss of mitochondrial transmembrane potential resulting in growth arrest, whereas membrane-bound PPIX induces a loss of membrane integrity and consequent necrosis. Although the absolute amount of intracellular photosensitizer concentration plays the main determining role for PDT efficiency, data indicate that intracellular localization has additional effects on the mode of cell damage.

5-aminolevulinic acid (ALA) mediated photodynamic therapy of bladder cancer cell lines

Topical application of 5-aminolevulinic acid (ALA) can be effectively used for photodynamic therapy and diagnosis of superficial bladder cancer. Administration of the heme precursor ALA leads to the selective accumulation of the photosensitizer protoporphyrin IX (PPIX) in certain types of tissue. The aim of this study was to determine the cellular PPIX concentration and the effect of photodynamic therapy mediated by ALA on two bladder cancer cell lines (RT4, J82) and a fibroblast cell line (N1). Following incubation with ALA the kinetics of cellular PPIX were examined using flow cytometry combined with extraction. The cancer cell lines showed considerably higher PPIX concentrations than the fibroblast cell line: RT4 1030, J82 710, and N1 110 ng PPIX/mg protein. Photodynamic therapy was performed with an incoherent light source (580 - 740 nm, 40 mW/cm2, 30 J/cm2). In contrast to the fibroblast cell line, which was resistant to photodynamic therapy, the cancer cell lines were effectively killed following the treatment as determined by MTT assay. This study suggests that ALA-mediated photodynamic therapy may be effective in transitional cell carcinoma of the bladder. Based on these findings, this therapeutic method should be further evaluated clinically.