Novel Targets of Protoporphyrin-IX Determined By Gene Expression Analysis

Abstract

Treatment with 5-Aminolevulinic acid-mediated photodynamic therapy is a promising therapeutic option for various carcinomas. An appropriate photosensitizer for photodynamic therapy is protoporphyrin-IX (PpIX), a light sensitive metabolite of heme synthesis. Incorporation of iron into PpIX leading to heme is carried out by Ferrochelatase (FECH). Earlier, we described a significant down regulation of FECH mRNA-expression and enzyme activity in carcinoma cells leading to an endogenous accumulation of PpIX. How PpIX affects the cell metabolism has not been examined so far. Thus, we tried to identify novel targets of PpIX with regard to cell proliferation, apoptosis and invasion. Endogenous generation of PpIX was induced by silencing of FECH in breast carcinoma MDA-MB-231 cells using a specific siRNA. Successful silencing of FECH was confirmed by RT-PCR and induction of PpIX was assessed by flow cytometry for each experiment. Subsequently, gene expression was determined using Affymetrix GeneChip® Human Gene 1.0 ST. Validation of microarray data was achieved by quantitative RT-PCR. Expression of one of the newly discovered target genes, BAMBI, was assessed by immunohistochemistry. In addition, the effect of silencing of FECH was examined by functional studies of cell apoptosis, invasion and wound healing. According to the gene expression analysis, an enhancement of PpIX suppressed Hedgehog as well as TGF-beta signaling. Expression of HHIP, a negative regulator of the hedgehog pathway, was found to be strongly increased after silencing of FECH. With regard to TGF-beta signaling, expression of the signaling inhibitor SMAD7 was strongly upregulated while the positive mediators SMAD2 and SMAD4 were less expressed after silencing FECH. Similarly, apoptosis of tumor cells was promoted, probably due to an increased expression of the pro-apoptotic gene APAF1 and a reduced expression of anti-apoptotic protein API5. Moreover, a significantly reduced invasion capability after treatment of cells with FECH siRNA was found. Here, we report that an accumulation of PpIX due to silencing of FECH affects various pathways and promotes apoptosis of tumor cells in different ways. Thus, silencing of FECH might have a tumor-suppressive effect. The search for substances which block FECH activity in a direct way therefore might be of high relevance for future cancer therapy approaches.