Novel Targets of Protoporphyrin-IX Determined By Gene Expression Analysis

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.


Introduction
Photodynamic therapy (PDT) is a promising approach for improved cancer treatment. PDT treatment has important advantages, because it is noninvasive with short treatment times, has a high repeatability without side effects, and shows little or no scar after healing. An appropriate photosensitizer for PDT is protoporphyrin IX (PpIX), a light sensitive metabolite of heme synthesis (1). Generation of PpIX depends on a sufficient intracellular delivery of the precursor substance aminolevulinic acid (ALA). Ferrochelatase FECH (EC 4.99.1.1) is responsible for the last step of heme-synthesis, incorporation of iron into PpIX leading to heme. After activation by light, PpIX induces the generation of reactive oxygen species responsible for cellular oxidation and subsequent tumor cell death. In previous studies, we found a significant down regulation of FECH mRNA-expression and enzyme activity in gastrointestinal carcinomas leading to an endogenous accumulation of PpIX within the tumor cells (2,3). Through inhibition of FECH mRNA expression using a specific siRNA-based probe we were able to amplify the specific endogenous fluorescence emission of PpIX within cancerous tissue. Successful induction of PpIX by silencing FECH was demonstrated e.g. by flow cytometry (4).
PpIX is also an activator of p53 and TAp73α tumor suppressors (5,6). According to the results of Zawacka-Pankau J et al. direct binding of PpIX to N-terminal regions of p53 and p73 can release these tumor suppressors from e.g. inhibition by MDM2. Reactivation of tumor suppressors might promote a massive cancer cell death after photodynamic treatment. Moreover, PpIX has been described to target tumor suppressors directly (7). Recently, Lee JM et al. have discovered that PpIX treatment is able to increase miR-199a-5p levels leading to the inhibition of various EMTtargets (8).
The aim of this paper was to identify novel targets of PpIX. Here, we present the first comprehensive gene expression study of the effects of an intracellular accumulation of PpIX in cancer cells. To this end, endogenous generation of PpIX was induced by treating breast carcinoma MDA-MB-231 cells with a specific FECH siRNA. Silencing of FECH was demonstrated by RT-PCR and successful induction of PpIX was assessed by flow cytometry for each experiment as described in (4). Subsequently, a microarray study of the gene expression was performed using Affymetrix GeneChip® Human Gene 1.0 ST. Validation of microarray data was achieved by quantitative RT-PCR of six genes. 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. The results show that an accumulation of PpIX due to silencing of FECH affects various pathways and promotes apoptosis of tumor cells in different ways. Moreover, we found a significantly reduced invasion capability after treatment of cells with FECH siRNA. In summary, intracellular accumulation of PpIX seems to have a tumor-suppressive effect.

Study Design and Gene Expression Analysis
Breast cancer cells MDA-MB-231 were divided into four treatment groups. For promoting the activity of heme metabolism, aminolevulinic acid (5-ALA) was added to all experimental groups except group D, which was a no treatment group. For silencing of FECH a specific siRNA described earlier was used (3). For comparison a control siRNA, Allstar siRNA, was applied. In each case, silencing of FECH was checked by RT-PCR and successful induction of PpIX was assessed by flow cytometry (see below). Gene expression was studied using Affymetrix GeneChip® Human Gene 1.0 ST.
Experimental details: Group A) are cells treated with 15 nM FECH siRNA + 0.1 mM 5-ALA; B) is a control group of cells which was treated with 15 nM Allstar siRNA + 0.1 mM 5-ALA; C) Cells were treated only with 0.1 mM 5-ALA but no siRNA; D) is an additional no treatment group. After treatment RNA was extracted as described below. The four treatment groups were analyzed three times in an independent fashion. Thus, twelve microarrays were examined. Preparation of labelled cRNA and hybridization was done according to the manufacturer's instructions using the Gene Chip Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, CA). Except the common quality control provided by gene chip operating software, a scientific workflow for chip quality control was set up in our lab including pseudo image plots, scatter MA plot and relative log expression plot. The resulting microarray data were preprocessed using the robust multichip average (RMA) algorithm via the BioConductor oligo package (9). Gene expression of FECH siRNA-treated cells was compared with that of the cells transfected with control siRNA by calculating the ratio of the signal intensity of FECH siRNAtransfected cells vs. the signal intensity of the controltransfected cells. A list of all the genes included in these microarrays and the normalized data have been deposited in the Gene Expression Omnibus database (http:// www.ncbi.nlm.nih.gov/geo/info/linking.html) under GEO accession number GSE135845.

Transient siRNA Transfection
All siRNA-molecules were designed according to standard procedures and obtained from Qiagen (Hilden, Germany). For silencing FECH the following sequence was used: siRNA FECH, 5'-gaauauccucuugguuccg - 3

RNA Extraction and RT-PCR
RNA extractions were carried out using RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Total RNA quality and yield were assessed using a bioanalyzer system (Agilent 2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA) and a spectrophotometer (NanoDrop ND-1000; NanoDrop Technologies, Wilmington, DE). Only RNA with an RNA Integrity Number (RIN) above 9.0 was used for further analysis. For quantification of the expression levels of various genes as found by microarray, RT-PCR was performed using pre-designed gene-specific TaqMan® probes and primer sets purchased from Applied Biosystems (Table 1). RT-PCR amplification was carried out using RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen) on an ABI PRISM 7900HT Sequence Detection System with fluorescence detection (Applied Biosystems), according to the manufacturer's instructions. Quantitative RT-PCR was run under the following conditions: 50°C for 15 minutes, 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. All samples were amplified in triplicate reactions. Gene expression was quantified relative to the expression of internal control ß-Actin using Sequence Detector Software (SDS 2.2, Applied Biosystems) and the relative quantification method for quantitative RT-PCR. In addition, ß-Actin levels were used to determine whether a given sample contained sufficient mRNA to be included in the study. Samples for which the Ct value for ß-Actin was above 22 were excluded from the analysis. The relative expression of each individual gene was calculated based on the determined ∆ CT values. The ∆ CT value is the difference between the Ct value for an individual gene and the internal reference control gene (13). ∆ CT values correspond to a log (-2) scale. The result of quantitative gene expression was calculated as a 2 -∆ CT relative to ß-Actin expression in the corresponding samples (14). Gene expression of FECH siRNA-treated cells was compared with that of the cells transfected with control siRNA by calculating the ratio of the relative amount of FECH siRNA-transfected cells vs. the relative amount of the control-transfected cells.  Table 1 For quantification of mRNA-expression, specific Assays-on-Demand, designed and produced by Applied Biosystems (Weiterstadt, Germany) were used.

Determination of Apoptosis of Breast Carcinoma Cells by Flow Cytometry
Apoptosis was determined using an Annexin V-FITC detection kit (BD Biosciences, CA) according to the manufacturer's instructions. For flow cytometric analysis, at least 10,000 MDA-MB-231 breast cancer cells were evaluated using a FACSCalibur (BD Biosciences, CA). Cell cycle distribution and pre-G1 fraction were determined in a quantitative way using the CellQUEST™ program. Annexin V positive-propidium iodide (PI) negative cells were counted as early apoptotic cells. All experiments were done at least in triplicates.

Detection of BAMBI Expression of Gastric Cancer Cells by Flow Cytometry
Harvest, wash the cells and adjust cell suspension to a concentration of 2.5x10 5 cells/ml in ice cold PBS, 10% FCS, 1% sodium azide. Add 2.5 µg/ml of primary antibody or isotype control antibody. Primary antibody for BAMBI detection was a mouse monoclonal (ab57043 from Abcam, Berlin, Germany). Isotype control was a monoclonal Mouse IgG1 (ab170190, Abcam). Cells were incubated overnight in the dark at 4°C. Cells were washed by centrifugation at 400 g for 7 min and resuspended in 100 µl ice cold PBS, 10% FCS, 1% sodium azide. Addition of secondary antibody (10 µg/ml) was followed by incubation 60 min at room temperature. Secondary antibody was a Goat Anti-Mouse IgG H&L-Alexa Fluor® 488 preadsorbed (ab150117, Abcam). Cells were washed by centrifugation at 400 g for 7 min and resuspended in 300 µl Stain-buffer (BD Biosciences, CA). For flow cytometric analysis, at least 10,000 cells were evaluated using a FACSCalibur ™ (BD Biosciences, CA).

Invasion Assay
Fibrosarcoma cells HT 1080 were treated with FECH siRNA as described above. Bio Coat Matrigel Invasion Chambers (Corning, USA) were used according to the instructions of the manufacturer. Briefly, 1x10 5 pretreated cells were seeded into the insert chambers, and the cells there were kept in DMEM medium without FKS. The wells around the chamber contained DMEM medium with 20% FKS. After an incubation period of 24 h at 37°C within in the CO 2 -incubator, non-invasive cells remaining within the chamber insert or invasive cells sitting at the bottom of the well were dissolved by careful treatment with the enzyme Accutase (Sigma-Aldrich, USA). Cells from each fraction were collected separately and cell number and viability were counted using a Cellometer Vision device and manually. As the last step, RNA was isolated and successful transfection with FECH siRNA was determined by quantitative PCR (see above).

Wound-Healing Assay
For wound healing assays, MDA-MB-231 cells treated with siFECH (see above) were used. Experiments were performed in triplicates using ibidi Culture-Inserts (ibidi, Munich, Germany). The inserts create a wound field with a defined gap of 9 mm x 9 mm for measuring the migratory and proliferation rates of cells. Migratory cells are able to extend protrusions and ultimately invade and close the wound field. Cells were seeded at a cell density of 5x10 5 cells/well in a 6-well plate and cultivated for 24 hours at 37°C and 5% CO 2 . One insert per well was transferred with sterile tweezers into a 24 well plate. The original cell solution was diluted to 1x10 6 cells per ml and 70 µl of the solution was added into each chamber of the insert. Wells were filled up with 1ml cell culture medium and plates were incubated overnight at 37°C and 5% CO 2 . Then the inserts were removed from the wells. Cell proliferation and migration after various incubation times was checked by microscopy.

Immunohistochemical Detection of BAMBI Expression
Immunohistochemistry was performed on 10 µm thin cryosections mounted on SuperFrost®Plus microscope slides (Menzel GmbH & Co KG, Braunschweig, Germany). MDA-MB-231 cells were treated as described above. Successful transfection with FECH siRNA was determined by quantitative PCR. Cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized with 0.1% PBS-Tween for 20 min. Cells were then incubated in 10% normal goat serum / 0.3M glycine in PBS to block non-specific protein-protein interactions. Primary antibody for BAMBI detection was a mouse monoclonal (ab57043, Abcam) (1µg/1x10 5 cells). Incubation was for 30 min at RT. Following washes in PBS, sections were incubated with secondary antibody Goat Anti-Mouse IgG H&L, Alexa Fluor® 488 preadsorbed (ab150117, Abcam) at 1/500 dilution for 30 min at RT. Microscopic pictures were taken using an inverted confocal laser scanning microscope Zeiss LSM710.

Statistical Analysis
Statistical calculation was done using GraphPad Prism version 5 for Windows (GraphPad Software).

Gene Expression of Heme Metabolism Enzymes After ALA-addition or Silencing of FECH
In a first approach the effects of silencing Ferrochelatase FECH (EC 4.99.1.1) on expression of the enzymes of heme biosynthesis was examined. FECH is responsible for the last step of heme-synthesis, incorporation of iron into PpIX. FECH down regulation and loss of enzymatic activity corresponds clearly with enhanced PpIX-dependent fluorescence (4). In addition, enhanced production of PpIX depends on a sufficient intracellular delivery of the precursor substance 5'aminolevulinic acid (5-ALA). Heme biosynthesis is controlled by the rate of the initial enzyme, mitochondrial 5'aminolevulinate Synthase 1 (ALAS1), which is under negative-feedback regulation by heme itself. Heme blocks transcription of the ALAS1 gene and stimulates the rate of degradation of the specific mRNA (15). Another essential enzyme in heme catabolism is heme oxygenase (HMOX). This enzyme cleaves heme to form biliverdin, carbon monoxide, and ferrous iron. Two forms have been described, an inducible heme oxygenase-1 HMOX1 and a constitutive heme oxygenase-2 HMOX2 (16). HMOX1 activity is induced by the substrate heme but also by various nonheme substances.
Effects of silencing FECH on gene expression in MDA-MB-231 cells were studied by microarray analysis. In order to promote PpIX generation, 5-ALA was added in all treatment groups except group D (no treatment group). The results of the microarray study showed that addition of 5-ALA vs. no treatment leads to a 4-fold increase in HMOX1 and a strong decrease of ALAS1 (Table 2, first column). The increased expression of HMOX1 and the decreased expression of ALAS1 is what one expects due to the increased synthesis of heme after addition of 5-ALA. Treatment of cells with FECH siRNA vs control siRNA decreased FECH expression strongly as determined by gene expression analysis ( Table 2, second column). Interestingly, also HMOX1 expression was reduced, probably due to the decrease in heme synthesis after silencing FECH. Thus, the results of the microarray analysis agree with the literature, confirming the quality of the microarray data.  Table 2 Differential expression of ALAS1, HMOX1 and FECH after addition of ALA in comparison to no treatment control (left side) or after silencing of FECH in comparison to Allstar control siRNA treatment (right side). Depicted are the ratios of the signal intensity as found in the microarray studies (see Material& Methods section).

Effects of Enhanced PpIX on the Expression of Genes Related to Apoptosis
Silencing of FECH led to an increased expression of the pro-apoptotic genes APAF1 and BAK1 in comparison with a treatment by control siRNA (Table 3). APAF1 encodes a cytoplasmic protein that initiates apoptosis (17). BAK1 facilitates the opening of the mitochondrial voltagedependent anion channel promoting a loss in membrane potential and the release of cytochrome c. BAK1 is also a target of p53 (18). In contrast, expression of anti-apoptotic proteins was reduced. API5 is an apoptosis inhibitory protein. API5 overexpression promotes tumor progression and suppresses apoptosis (19). In agreement, expression of the pro-apoptotic gene CASP6 was decreased. These data suggest that enhanced PpIX due to silencing FECH promotes apoptosis in several ways.

TGF-beta Pathway Blocked by Increased PpIX Due to Silencing of FECH
According to the literature, TGF-beta is thought to be a tumor suppressor (20). TGF-beta signaling prevents growth by inducing cell cycle arrest and apoptosis through repression of cyclin dependent kinases and mitogenic c-MYC in normal epithelia and in early tumorigenic cells. However, in late stage tumorigenic cells TGF-beta stimulates cancer progression and metastasis e.g. by activation of epithelial-tomesenchymal transition (EMT) (21). According to the microarray data, enhanced PpIX due to silencing FECH blocks TGF-beta signaling (Table 4). On the one hand, the inhibitory mediator SMAD7 was strongly upregulated. On the other hand, positive mediators SMAD2 and SMAD4 were less expressed. Upregulation of SMAD7 expression and reduced expression of TGFBR1 was confirmed by RT-PCR (Table 6). SMAD proteins are intracellular mediators of TGF-beta superfamily. SMAD7 inhibits TGF-beta signaling. Similarly, a reduced expression of CRIM1 was detected. CRIM1 promotes tumor development and progression through its potential role in EMT (22). Moreover, an enhanced expression of BAMBI (BMP and activin membrane-bound inhibitor homolog) was found by gene expression. Bambi overexpression was confirmed on the protein level by immunohistochemistry for breast carcinoma cells MDA-MB-231 and by flow cytometry for gastric carcinoma cells MKN28 (Figures 1 and 2). Recently, BAMBI overexpression was found to inhibit growth and metastasis of gastric cancer cells (23). Thus, our data suggest that PpIX reduces TGF-beta signaling and tumor cell growth.  FACS staining showed that BAMBI is highly expressed on MKN-28 cells treated with FECH siRNA. Abbreviations: TRF w/o Ab, transfection reagent, no antibody; control w/o Ab, control siRNA, no antibody; FECH w/o Ab, FECH siRNA, no antibody. TRF sec Ab, transfection reagent, secondary antibody; control sec Ab, control siRNA, secondary antibody; FECH sec Ab, FECH siRNA, secondary antibody. TRF iso Ab, transfection reagent, isotype antibody; control iso Ab, control siRNA, isotype antibody; FECH iso Ab, FECH siRNA, isotype antibody. TRF BAMBI, transfection reagent, BAMBI antibody; control BAMBI, control siRNA, BAMBI antibody; FECH BAMBI, FECH siRNA, BAMBI antibody.

Hedgehog Signaling Blocked by Increased PpIX Due to Silencing of FECH
Hedgehog (HH) signaling affects cell differentiation, cell proliferation and tissue polarity, as well as the maintenance of stem cells, tissue repair, and regeneration (24). Three ligands, Indian, Sonic, and Desert HH activate this pathway. Binding of HH ligands leads to nuclear translocation of GLI transcription factors. According to gene expression analysis, silencing of FECH promoted HHIP expression (Table 5).
HHIP overexpression was confirmed PCR (Table 6). HHIP functions as a negative regulator of the hedgehog pathway. Meanwhile, it has been found that HHIP-overexpression inhibits gastric carcinoma cell proliferation, migration and invasion (25). Similarly, expression of Glioma-associated oncogene 3 (Gli3) was found to be increased by microarray data as well as by PCR. GLi3 acts as the main repressor of the pathway in the absence of HH ligands. Thus, silencing of FECH seems to block Hedgehog signaling as well as TGFbeta signaling as shown above.

Other Genes of Interest
Interestingly, silencing of FECH affects also WNT signaling. Lee JM et al. found that PpIX treatment increased miR-199a-5p, leading to a decreased expression of various EMT-targets (8). This micro-RNA was found to affect multiple biological processes such as cell proliferation, apoptosis, migration and invasion (26). In addition, Kim BK et al. found that miR-199a-5p interacts with Wnt/β-catenin pathway and reduces Frizzled Class Receptor 6 (FZD6) expression (27). In agreement, we found a strong downregulation of FZD6 (ratio 0.35) after silencing of FECH. This is another hint that silencing FECH/ enhanced PpIX exerts a tumor-suppressive effect. However, also an increased expression (2.05) of Cyclin D2 (CCND2) was found. CCND2 belongs to the D-type cyclin family and participates in cell cycle progression and the induction of cancer progression (28). Thus, overexpression of CCND2 as found here might have a tumor-promoting effect.
As mentioned in the introduction, Joanna Zawacka-Pankau et al. (6) have shown that PpIX induces both p53-dependent and -independent apoptosis since PpIX is an activator of p53 and TAp73α tumor suppressors. Involved in this process is early growth response 2 (EGR2) which was identified as a direct transcriptional target of p53 family and which is able to induce the p53-dependent apoptotic pathway (29). In agreement, here we found that EGR2 is strongly enhanced after silencing of FECH (ratio 2.12) and might be activated by PpIX directly.

Functional Studies
The results of the microarray study support the notion that an enhanced amount of PpIX promotes apoptosis of cancer cells (Table 2). Accordingly, the functional studies showed an increase of early apoptosis of MDA-MB-231 breast after FECH siRNA treatment ( Figure 3) in comparison to cells treated with control siRNA. According to the gene expression analysis, enhanced PpIX suppresses Hedgehog as well as TGF-beta signaling. Moreover, expression of BAMBI (BMP and activin membrane-bound inhibitor homolog) which negatively regulates TGF-beta signaling was found to be enhanced. In agreement, in functional studies of MDA-MB-231 breast cancer cells treated with FECH siRNA a reduced capacity of the cells for invasion ( Figure 4) and a slightly delayed capacity for wound healing was demonstrated ( Figure 5).  Abbreviations: NTC, no treatment group; TRF, Transfection reagent only; Control, treated with Allstar siRNA; FECH, treated with FECH siRNA (Significance p<0.001, t-Test of FECH vs. control siRNA). Apoptosis was determined using an Annexin V-FITC detection kit (BD Biosciences) according to the manufacturer's instructions. For flow cytometric analysis, at least 10,000 cells were evaluated using a FACSCalibur. Cell cycle distribution and pre-G1 fraction were determined in a quantitative way using the CellQUEST™ program. Annexin V positive -PI negative cells were counted as early apoptotic cells. All experiments were done in triplicates.

Figure 4. Reduced invasion of cells after treatment with FECH siRNA.
Treatment with FECH siRNA significantly (p<0.02) reduced the invasive capacity of cancer cells. Invasion was examined as described in Materials& Methods section. Briefly, Matrigel Invasion Chambers (Corning, USA) were used according to the instructions of the manufacturer. Briefly, 1x10 5 cells were seeded into the insert chambers, and the cells there were kept in DMEM medium without FKS. The wells around the chamber contained DMEM medium with 20% FKS. After an incubation period of 24 h at 37°C within in the CO2-incubator, non-invasive cells remaining within the chamber insert or invasive cells sitting at the bottom of the well were dissolved by careful treatment by enzyme treatment. Cells from each fraction were collected separately and cell number and viability. As the last step, RNA was isolated and successful transfection with FECH siRNA was determined by quantitative PCR. Representative images were taken at 8 hours post-scratch. Wound healing assays were performed four times. Wound healing of cells treated with FECH siRNA was delayed, but significance was not reached. Wound healing was examined as described in Materials& Methods section. Abbreviations: NTC, no treatment group; TRF, Transfection reagent only; Control, treated with Allstar siRNA; FECH, treated with FECH siRNA. Table 6 Gene expression of FECH siRNA-treated breast carcinoma cells MDA-MB-231 cells was compared with that of cells transfected with Allstar control siRNA by calculating the ratio of the relative amount of FECH siRNA-transfected cells vs. the relative amount of the control-transfected cells (Material& Methods section). FECH vs Control siRNA: Fold change of expression after silencing FECH by specific siRNA in comparison to Allstar siRNA.

Conclusion
The aim of this study was the generation of a database that allows the identification of genes or groups of genes, which show an altered expression after increasing PpIX intracellularly. In contrast to other investigations, PpIX was not added to the cell culture medium directly, however the concentration of endogenous PpIX was increased through silencing of FECH. This increase was confirmed for each experiment by flow cytometry of fluorescent PpIX. An enhanced expression of PpIX due to a silencing of FECH seems to affect the properties of cancer cells in several ways. Both gene expression analysis and functional assays suggest that an enhanced amount of PpIX promotes apoptosis of cancer cells. Moreover, according to the microarray results, HHIP and SMAD7 are important effector molecules for the modified functional characteristics of cells treated with FECH siRNA. Future studies will show how PpIX affects apoptosis and invasion by controlling gene expression of these mediator molecules. Another important aspect is that an enhanced amount of PpIX seems to have a tumor-protective or suppressive effect on breast cancer cells. Therefore, the search for substances that block FECH activity directly might be of considerable relevance for future cancer therapy approaches.