RNA expression profiling of normal and tumor cells following photodynamic therapy with 5-aminolevulinic acid-induced protoporphyrin IX in vitro.
Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism of tumor phototoxicity is important to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with cell line-specific LD50 doses of light after exposure to 5-aminolevulinic acid (100 microg/mL), and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The RNA was hybridized to the metg001A Affymetrix GeneChip containing 2,800 genes, focusing on cancer-related and growth regulatory targets. Comparing the gene expression profiles between the different samples, 40 genes (e.g., SOD2, LUC7A, CASP8, and DUSP1) were identified as significantly altered in comparison with the control samples, and grouped according to their gene ontology. We selected caspase-8 (CASP8) and dual specificity phosphatase 1 (DUSP1) for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative reverse transcription-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. In contrast, FOS RNA was found continuously up-regulated over time in all three cell lines. Induction of DUSP1 protein expression was clearly shown after 1 hour using Western blot analysis. Interestingly, no changes of caspase-8 protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. According to caspase-8, the active caspase 3 fragment was found only in the normal urothelial cell line (UROtsa) 1 hour after photodynamic therapy. Combined data analysis suggests that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. RNA expression profiling of normal and tumor cell lines following photodynamic therapy with 5-aminolevulinic acid gave insight into the major molecular mechanisms induced by photodynamic therapy.
Wild
Peter J
PJ
Institute of Pathology, University of Regensburg, Regensburg, Germany.
Krieg
Rene C
RC
Seidl
Juergen
J
Stoehr
Robert
R
Reher
Kerstin
K
Hofmann
Claudia
C
Louhelainen
Jari
J
Rosenthal
André
A
Hartmann
Arndt
A
Pilarsky
Christian
C
Bosserhoff
Anja K
AK
Knuechel
Ruth
R
D000622:Aminolevulinic Acid / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D016000:Cluster Analysis; D004305:Dose-Response Relationship, Drug; D054638:Dual Specificity Phosphatase 1; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D005821:Genetic Techniques; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D008928:Mitochondria / Q000378:metabolism; D008957:Models, Genetic; D015233:Models, Statistical; D009336:Necrosis; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010778:Photochemotherapy / Q000379:methods*; D017319:Photosensitizing Agents / Q000494:pharmacology; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011524:Protoporphyrins / Q000235:genetics*; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D017319:Photosensitizing Agents; D011524:Protoporphyrins; D012313:RNA; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases