The FASEB Journal • Research Communication
Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation in colon cancer Wolfgang Kemmner,*,1 Kayiu Wan,* Steffen Ru¨ttinger,† Bernd Ebert,† Rainer Macdonald,† Ursula Klamm,‡ and K. Thomas Moesta‡ *Max Delbrueck Center for Molecular Medicine, Berlin, Germany; †Physikalisch-Technische Bundesanstalt, Department of Biomedical Optics, Berlin, Germany; and ‡Robert Roessle Clinic, Clinic for Surgical Oncology, Charite–Universita¨tsmedizin Berlin, Berlin, Germany Hemes and heme proteins are vital components of essentially every cell of virtually every eukaryote organism. Previously, we demonstrated accumulation of the heme precursor protoporphyrin-IX (PpIX) in gastrointestinal tumor tissues. To elucidate the mechanisms of PpIX accumulation by quantitative reverse transcriptase-polymerase chain reaction (RTPCR), we studied expression of the relevant enzymes of the heme synthetic pathway. Here, we describe a significant down-regulation of ferrochelatase (FECH) mRNA expression in gastric, colonic, and rectal carcinomas. Accordingly, in an in vitro model of several carcinoma cell lines, ferrochelatase down-regulation and loss of enzymatic activity corresponded with an enhanced PpIX-dependent fluorescence. Direct detection of PpIX in minute amounts was achieved by a specifically developed pulsed solid-state laser dual delay fluorimetry setup. Silencing of FECH using small interfering RNA (siRNA) technology led to a maximum 50-fold increased PpIX accumulation, imageable by a specifically adapted two-photon microscopy unit. Our results show that in malignant tissue a transcriptional down-regulation of FECH occurs, which causes endogenous PpIX accumulation. Furthermore, accumulation of intracellular PpIX because of FECH siRNA silencing provides a small-molecule-based approach to molecular imaging and molecular therapy—Kemmner, W., Wan, K., Ru¨ttinger, S., Ebert, B., Macdonald, R., Klamm, U., Moesta, K. T. Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation in colon cancer. FASEB J. 22, 500 –509 (2008)
ABSTRACT
Key Words: heme metabolism 䡠 gastrointestinal carcinomas 䡠 siRNA silencing, 䡠 cytoplasmatic red fluorescence
Hemes and heme proteins are vital components of essentially every cell of virtually every eukaryote organism (1). All nucleated human cells require at least small quantities of heme, which is essential for the cellular energy metabolism. Thus, the enzymes of the heme synthetic pathway are expressed ubiquitously. In the first step of heme synthesis, 5-aminolevulinic acid (ALA) is formed from glycine and succinyl coenzyme A by ALA synthase (ALA-S1; EC 2.3.1.37). Subsequently, 500
porphobilinogen deaminase (PBG-D; EC 4.3.1.8) catalyzes the formation of uroporphyrinogen from porphobilinogen. The last step is the incorporation of iron into protoporphyrin IX (PpIX), by ferrochelatase (FECH; EC 4.99.1.1), which is localized within the inner mitochondrial membrane. Degradation of heme is catalyzed by heme oxygenase (HO; EC 1.14.99.3). The omnipresence of the heme synthetic pathway currently is exploited by a number of research groups to induce substantial quantities of protoporphyrin IX, the heme immediately preceding substrate, by administration of 5-ALA, bypassing the negative feedback control on ALA-S1 synthase. PpIX, a fluorescing and photodynamically active chromophore, has been shown useful for detection and treatment of neoplastic diseases (2– 4). While the general mechanism of PpIX accumulation is well known, it is not understood why this accumulation is specific for neoplastic tissues. The possibility that a loss of FECH activity might be responsible for enhanced PpIX accumulation in human carcinomas had been proposed in 1984 by Dailey et al. (5). In this paper, the researchers state that the low FECH activity of cancerous cells “may provide one means whereby some porphyrins accumulate in tumors.” van Hillegersberg et al. (6) showed that oral administration of 5-ALA resulted in progressive accumulation of protoporphyrin in a rat colon carcinoma but not in the surrounding liver tissue. They suggested that the selective accumulation of porphyrins likely is caused by a relative FECH deficiency in malignant tissue. On the contrary, Hua et al. (7) did not find a direct relationship between the level of activities of the enzymes involved in heme biosynthesis and the amount of PpIX formed in rat mammary adenocarcinomas. Hinnen et al. (8) suggested that, after ALA-administration, porphyrins would accumulate because of an imbalance between PBG-D and FECH activities. Investigations involving in vitro cultures of urothelial cells and fibroblasts (9) showed that the most important metabolic step for PpIX accumulation is the transition from PpIX to 1
Correspondence: Max Delbrueck Center for Molecular Medicine, Robert Roessle Str 10, 13125 Berlin, Germany. E-mail:
[email protected] doi: 10.1096/fj.07-8888com 0892-6638/08/0022-0500 © FASEB
heme, which is controlled by FECH activity and intracellular iron. Previously, we demonstrated that certain gastrointestinal tumor tissues accumulate PpIX even in the absence of exogenously administered ALA (10). To elucidate the mechanisms of PpIX accumulation in cancer tissue, we studied the quantitative expression and activity of relevant enzymes of the heme synthetic pathway in both gastrointestinal carcinomas and in an in vitro model and used small interfering RNA (siRNA) technology to knock down FECH expression.
Cell culture MDA-MB-435 cells, originally derived from a mammary carcinoma, and LS174T, SW480, and HT29, colorectal cancer cell lines originating from the American Type Culture Collection, were grown in DMEM supplemented with 10% FBS, 2 mM glutamine, 0.6 g penicillin/ml (equivalent to 100 U) and 0.1 mg streptomycin/ml medium. Subconfluent adherent cells were harvested by a mixture of trypsin (0.05%) and EDTA (0.02%), rescued with their own medium, and washed with PBS. Tissue preparation and RNA extraction
MATERIALS AND METHODS Patients and tissue samples Patients had been treated in the Department of Surgery and Surgical Oncology, Robert-Roessle-Klinik, in Berlin, Germany. Carcinoma tissues were excised carefully from tumor specimens. Nonmalignant mucosa also was scraped off at a distance of more than 50 mm from the tumor margin. Tissue was snap-frozen in liquid nitrogen by the tumor bank service of the Robert-Roessle-Klinik. Carcinomas were classified and characterized according to the World Health Organization’s UICC (Union Internationale Contre le Cancer) system. Normal tissue refers to peritumoral tissue verified to be free of any pathological traits. All patients had been treated by surgery between 1999 and 2001. In each case, a comprehensive histopathological and clinical record is available. Patient specimens were collected after informed consent and used only after ethics committee approval (Charite´, Berlin). For quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) studies, a panel of 18 cases with esophageal carcinomas, 20 cases with gastric carcinomas, 15 cases with colonic carcinomas, and 15 cases with rectal carcinomas, along with their nonmalignant counterparts, was examined. Tumor stages were equally represented. Materials and chemicals Taqman 7000 SDS Cycler, DEPC-water and all real-time PCR chemicals, unless otherwise stated, were from Applied Biosystems (Weiterstadt, Germany). Agarose was obtained from BioWhittaker Molecular Applications (Rockland, ME, USA). Trizol LS reagent, guanidine thiocyanate-phenol-chloroform, ethidium bromide, Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum, PBS, antibiotics, and glutamine were purchased from Gibco Life Technologies (Eggenstein, Germany). Plasmid pcDNA3.1/V5-His-TOPO, pcDNA4/HisMax-TOPO), and TOP10 Escherichia coli were obtained from Invitrogen (Karlsruhe, Germany), RiboGreen RNA quantification reagent from Molecular Probes (Leiden, Netherlands), and BSA fraction 5 and glutamine from PAA (Co¨lbe, Germany). M-MLV reverse transcriptase, RNasin, RNaseH, and dNTP were purchased from ProMega (Mannheim, Germany) and siRNA, transfection messenger reagent, RNeasy RNA-extraction kit, QIAshredder kit, One-Step RT PCR kit, and QiaEx-II gel extraction kit from Qiagen (Hilden, Germany). Phenol/chloroform and all other chemicals were purchased from Roth (Karlsruhe, Germany). Sucrose was obtained from Serva (Heidelberg, Germany). Protoporphyrin IX disodium salt and protoporphyrin IX zinc (II), Triton X-100, linear polyacrylamide (GenElute LPA), BSA, Mayer’s hematoxylin solution, and other standard chemicals were obtained from Sigma-Aldrich (Munich, Germany). PROTOPORPHYRIN-IX ACCUMULATION IN COLON CANCER
Cryosections from colorectal and normal tissue specimens were transferred into RLT buffer containing 140 M -mercaptoethanol and frozen at – 80°C. To ensure absence of tumor cells in normal tissue and sufficient tumor content in cancer specimens, all tissue specimens were evaluated by a pathologist. Only specimens containing more than 60% epithelial cells but no Peyer’s patches or necrotic areas were further processed. RNA was isolated using QIAshredder and RNeasy kits. Integrity of isolated mRNA was checked by -Actin One-Step RT PCR, and the RNA concentration was determined photometrically. About 3 g RNA was used for cDNA synthesis. In the presence of 4 M random hexamer primer, RNA was incubated for 10 min at 70°C, then the mixture was kept on ice for another 10 min. After addition of M-MLV buffer, 8 U/l M-MLV reverse transcriptase, 0.8 U/l RNasin, 0.1 g/l BSA and 1.25 mM dNTP and 1 h incubation at 37°C, twice the volume of absolute ethanol was added. Following 30 min at – 40°C, samples were spun down in a centrifuge at 15,000 g at 4°C for 20 min and washed once with 70% ethanol. Finally, cDNA was reconstituted in 50 l DEPC water and frozen at –20°C. Quantitative real-time Taqman PCR For all genes, Assays-on-Demand (ABI) primer and probes were employed. Though sequences are kept confidential by the manufacturer, in order to obtain accurate sequence information, the PCR product was inserted into plasmid pcDNA3.1/V5-His-TOPO or pcDNA4/His-Max-TOPO (Invitrogen), amplified in TOP10 E. coli, and sequenced using the plasmid-specific primer T7: 5⬘-TAA TAC GAC TCA CTA TAG GG-3⬘. Accordingly, the sequence of -actin PCR product amplified by the ABI primers is 5⬘-CCT GAA CCCCAA GGC CAA CCG CGA GAA GAT GAC CCA GAT CAT GTT TGA GAC CTT TAA CAC CCC AGC CAT TAC GTT GCT ATC CTG TCT GTA AGG-3⬘, and by HPRT is 5⬘-TTG GTC AGG CAG TAT AAT CCA AAG ATG GTC AAG GTC GCA AGC TTG CTG GTG AAA AGG ACC CCA CGA AGT GTT GGA TAT AAG CCA GAC TTT GTT GGAT TTG AAA-3⬘. The -actin sequence is identical to that of Homo sapiens actin, beta (ACTB), mRNA; GenBank accession NM_001101. The hypoxanthinphosphoribosyltransferase (HPRT) sequence is identical to that of Homo sapiens HPRT1 (Lesch-Nyhan syndrome), mRNA; GenBank accession NM_000194. Blast analysis showed that the primers used for the reaction are intron-spanning primers, which amplify only cDNA reverse transcribed from mRNA but not genomic DNA. For quantitative analysis of gene expression, 100 ng cDNA was used per well. All reactions were carried out in triplicate. After mixing with the appropriate volume of TaqMan Universal PCR Master Mix, quantitative real-time PCR was run in a MicroAmp Optical 96-Well Reaction Plate using the ABI 7000 Sequence Detection System. Thermal cycle conditions were as 501
follows: 95°C for 10 min initially, then 40 cycles of 95°C for 15 s and 60°C for 1 min. To analyze expression data according to the ⌬⌬Ct method (11), Ct values were exported from the ABI Prism 7000 SDS software into Microsoft Excel (Seattle, WA, USA). Ct values of a cDNA stock from the cell line LS174T were used as calibrator. Thereby, gene expression in a sample under investigation is reported as a multiple of cell line expression. This expression is achieved by employing the equation Relative amount ⫽ 2 ⫺(⌬Ct sample ⫺ ⌬Ct calibrator) wherein ⌬Ct stands for the difference between Ct values of gene of interest and housekeeper -actin. PpIX determination PpIX fluorescence in homogenized centrifuged pellets from cell culture was quantitatively determined by time-resolved fluorescence spectroscopy. PpIX fluorescence was excited with an optical parametric oscillator (OPO, GWU-Lasertechnik, Erftstadt, Germany), pumped by the third harmonic (⫽355 nm, Epulse⫽100 mJ) of a Q-switched Nd:YAG laser (GCR-230, Spectra-Physics Inc., Mountain View, CA, USA). The OPO was tuned to provide laser radiation at ⫽ 505 nm to excite PpIX fluorescence. The energy and duration of output pulses amounted typically to 50 J and 3 ns, respectively. The laser beam was coupled into a 600-m hard-clad silica fiber used for illumination of cell samples. The fluorescence light was collected by the same fiber, and a dichroic beam splitter served to separate reflected laser light from fluorescence. About 5% of the remaining fluorescence intensity was separated by a quartz plate and coupled into a hard-clad silica fiber, ⬃5 m long, whereas the main fluorescence intensity (90%) was focused into a second fiber, 1 m long. In this way, fluorescence in the first channel was delayed with respect to the second channel by ⬃20 ns, allowing the recording of prompt and delayed fluorescence intensity simultaneously. For this purpose, the ends of both fibers were attached, one above the other, to the entrance slit of an imaging polychromator equipped with an intensified CCDcamera (Model ICCD-576, Princeton Instruments, Inc., Trenton, NJ, USA). The intensifier was gated by an electrical pulse (⫺180 V) of ⬃10 ns duration derived from a high-voltage (HV) pulse generator synchronized with the laser pulse. The HV pulse generator was triggered by a pulse provided by the power supply of the Nd:YAG laser and appropriately delayed by means of a digital delay generator. In this way, immediate and delayed fluorescence intensities were recorded. Furthermore, two long pass filters were used (50%⫽550 nm) to cut off the remaining scattered excitation light from emitted fluorescence light. For quantification, we calculated the normalized fluorescence intensity of the main PpIX fluorescence band at ⫽ 633 nm. A factor of 18 corresponds to the ratio of the transmitted and reflected fluorescence intensity by the quartz plate. Two-photon excitation microscopy Experiments were carried out on two-photon excitation confocal equipment established in our institute by utilizing an inverted microscope stand (Zeiss Axiovert 35M, Carl Zeiss, Oberkochen, Germany). In short, the system consists of a mode-locked titanium:saphire laser (MaiTai, Spectra-Physics) that generates pulses with a duration of ⬃100 fs at a repetition rate of 80 MHz. After passing beam neutral density filters and a beam expander, the excitation light is directed to a dichroic mirror that is highly reflective in the near infrared and transparent below 725 nm (725DCX SP, AHF, Germany). 502
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Thus, the laser beam is coupled into the optical epi-illumination path of the microscope. To allow imaging, we incorporated a two-dimensional laser beam scanner consisting of two mirrors driven by a closed loop galvanometer scanner (GSI Lumonics, Moorpark, CA, USA). A tube lens and a ⫻10 ocular (both Carl Zeiss) act as a relay lens system between the scanner mirrors and objective. Finally, the excitation light is focused into the sample by a ⫻63 C-apochromat, water immersion objective (N.A. 1.2, Carl Zeiss). The fluorescence radiation is collected by the same microscope objective and then passes scan lenses and scanning mirrors. Since for two-photon excitation the excited fluorescence light is of shorter wavelength than the excitation light, the generated fluorescence signal passes the dichroic beam-splitter mentioned above. To further suppress scattered excitation light, an additional short pass (700 SP, AHF) was incorporated into the detection path. Although no confocal aperture is needed for axial sectioning in two-photon microscopy, its inclusion in the detection path reduces the contribution of scattered out-of-focus fluorescence and residual ambient light, thereby increasing the signal/background ratio. Therefore, the fluorescence intensity was imaged by an achromat (f⫽600 mm) onto an adjustable aperture, the diameter of which was set to 1.8 mm. The overall magnification was measured to be ⬃1530 for the ⫻63 magnification objective. Light passing the confocal aperture was spectrally filtered for analysis by a long pass filter with 50% transmission at 595 nm and two band pass filters (535⫾17.5) nm and (635⫾20) nm, (AHF Analysentechnik AG, Tuebingen, Germany). The light of each path was focused onto an avalanche photo detector (SPCM-AQR-14, Perkin Elmer, Inc., Santa Clara, CA, USA), which converts the detected photons into standard TTL pulses. The pulses are counted by a PC plug-in counter board (pci-6602, National Instruments, Munich, Germany). Measurement of excitation spectra (not shown) in the tunable range of the excitation laser (760 –920 nm) yielded a PpIX excitation maximum at ⫽ 760 nm. The one-photon excitation maximum of PpIX is located at ⬃400 nm. As expected (12), the two-photon excitation maximum is slightly blue-shifted compared to the one-photon excitation spectrum. Images of 200 ⫻ 200 pixels were acquired by line scans at ⫽ 760 nm. The excitation power measured at the sample position was 0.8 ⫾ 0.1 mW. The interpixel distance selected amounted to 0.98 m, corresponding to a total field of view of 197 ⫻ 197 m. The exposure time per pixel was varied between 0.25 ms and 1.25 ms, according to the overall fluorescence intensity of the sample, to ensure good detection statistics with the lowest possible excitation power. Total data acquisition times, therefore, varied between 10 and 50 s. Determination of FECH enzyme activity FECH activity was determined as described by van Hillegersberg et al. (6) with minor modifications. About 5 ⫻ 107 frozen cells were powdered in 500 l ice-cold H2O using a tissue homogenizer (Braun, Melsungen, Germany). Then, 50 l of the homogenate was added to 100 l buffer A, consisting of 25 mM Tris/HCl, 0.1% Triton X-100, 1.75 mM palmitic acid, pH 8.2. Protein content in the supernatant was determined using the Bio-Rad protein assay. Fifty l of a 250 M protoporphyrin solution in 0.01 N KOH was added, and the reaction was started by addition of 50 l 200 M zinc acetate. After 60 min at 37°C, the reaction was stopped by addition of 1 ml dimethyl sulfoxide:methanol (30:70). After centrifugation at 16,000 g for 5 min, 100 l supernatant was injected into a reverse-phase RP18 column HPLC (Shimadzu, Duisburg, Germany) with acetone/methanol/water/formic acid (560:240:200:2), 1 ml/min as the mobile phase. Zinc-proto-
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porphyrin was detected using a fluorimeter (Merck, Darmstadt, Germany) with an excitation ⫽ 415 nm and an emission wavelength ⫽ 580 nm. One unit of activity was defined as the amount of enzyme catalyzing the formation of 1 mol product/min. The activity of the enzyme is given in nU/mg protein.
Statistical analysis Correlations between clinical parameters and gene expression as measured by qPCR were analyzed by Wilcoxon test. A value of P ⬍ 0.05 was considered significant. All qPCR data were analyzed with SPSS 14.0 (SPSS, Chicago, IL, USA).
Transient siRNA transfection All siRNA molecules were designed according to standard procedures and obtained from Qiagen. Three different sequences were used: siRNA sequence FECH-510, 5⬘-GAUUCAAGAGCAGUACCGC-3⬘, covering the FECH mRNA, transcript variant 1, with accession NM_001012515.1 between nucleotides 510 –528, and FECH mRNA, transcript variant 2, with accession NM_000140.2 between nucleotides 492–510, respectively; siRNA FECH-1140, 5⬘-GAAUAUCCUCUUGGUUCCG-3⬘, covering the FECH mRNA, transcript variant 1, with accession NM_001012515.1 between nucleotides 1140 – 1158, and FECH mRNA, transcript variant 2, with accession NM_000140.2 between nucleotides 1122–1140, respectively; siRNA FECH-2107, 5⬘-GUACAGUGUUCAUGAUACG-3⬘, covering the FECH mRNA, transcript variant 1, with accession NM_001012515.1 between nucleotides 2107–2125, and FECH mRNA, transcript variant 2, with accession NM_000140.2 between nucleotides 2089 –2107, respectively. Cell seeding density in 6-well plates was always 2 ⫻ 105 cells per well. The first siRNA transfection started at day 2 after seeding. Briefly, siRNA in a concentration of 0.1 g/l was mixed with buffer and enhancer and incubated at room temperature for 5 min (total volume 110 l). Thereafter, 2.2 l TransMessenger reagent was added, and the whole solution was mixed by pipetting up and down. Then, the samples were incubated for 10 min at room temperature to allow transfection-complex formation to take place. During this time, the growth medium was gently aspirated from the cell dish and cells were carefully washed. DMEM medium (without serum or antibiotics), 600 l, was added into the nosiRNA and no-transfection reagent control wells, and 500 l/well DMEM medium into the other wells. Then, 100 l of the transfection complex was added into the corresponding wells. Cells were incubated with the transfection complexes for 4 h under their normal growth conditions, and then 1000 l DMEM medium containing serum and antibiotics was added. A second siRNA treatment was conducted on day 5. Cells were harvested at day 6. All experiments were done at least in triplicate.
RESULTS Expression of heme metabolism enzymes and HO in colorectal carcinomas ALA-S1, PBG-D, FECH, and HO mRNA expression were determined in microdissected specimens of colorectal carcinoma tissue and corresponding normal mucosa. Quantitative single-step multiplex RT-PCR in relation to that of the housekeeper gene -actin and in comparison with the colorectal cell line SW480 was used (Fig. 1). Results show that ALA-S1 mRNA expression was reduced in colorectal carcinomas compared to that of normal tissue of the same patients (P⬍0.031, Wilcoxon-test of matched pairs). No significant alterations in PBG-D mRNA expression between colorectal carcinomas and normal mucosa were detected. HO mRNA expression was reduced in colorectal carcinomas compared to normal tissue of the same patients (P⬍0.021, Wilcoxon test of matched pairs). Similarly, FECH expression was significantly reduced in colorectal carcinomas (see below). FECH mRNA expression in microdissected specimens of gastrointestinal carcinomas FECH mRNA expression in microdissected samples of gastrointestinal tissue was determined by quantitative single-step multiplex RT-PCR as described above. In a second series of experiments, FECH expression in colonic and rectal carcinomas was determined in relation to another housekeeping gene, namely HPRT, by quantitative RT-PCR as well (data not shown). The data
Figure 1. Expression of heme metabolism enzymes in colorectal carcinomas. Determination of mRNA expression of ALAS, HO, PBGD, and FC in colorectal tumor tissue or corresponding normal mucosa using quantitative RT-PCR. A total of 30 cases of colorectal carcinomas have been examined for each enzyme. FECHmRNA expression was significantly higher in normal mucosa compared to carcinoma tissue (P⬍0.005, Wilcoxon test). Computational analysis was done using the ⌬⌬Ct method with reference to ß-actin expression (see Materials and Methods). Abbreviations: CA, carcinoma tissue; NM, normal mucosa. x-axis: tissue samples; y-axis, relative amount of expression; *,°samples rated as extremes or outliers. PROTOPORPHYRIN-IX ACCUMULATION IN COLON CANCER
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of this second experiment are in good agreement with results obtained using -actin. In general, FECH expression is significantly reduced in gastrointestinal carcinomas in comparison to normal mucosa of the same patient. This decrease in FECH mRNA expression was highly significant as well in gastric (P⬍0.001), colonic (P⬍0.005), and rectal (P⬍0.031) carcinomas compared to corresponding normal mucosa (Fig. 2, Wilcoxon test of matched pairs). FECH expression of esophageal carcinomas also was lower than that of corresponding normal mucosa. However, this difference was not significant, presumably because FECH expression in esophageal tissues showed higher variation. FECH mRNA expression of cell lines correlates with their potential to accumulate PpIX after ALA stimulation In an in vitro model consisting of several carcinoma lines, FECH mRNA expression and FECH enzyme activity were independently determined. FECH mRNA expression was measured by quantitative single-step multiplex RT-PCR in relation to that of -actin. FECH enzyme activity was determined by using zinc-protoporphyrin as substrate as described by van Hillegersberg et al. (6). FECH mRNA expression correlated well with enzyme activity (Table 1). PpIX accumulation was induced by exogenous ALA stimulation with 0.1 mM ALA for 4 h and determined by time-delayed fluorescence measurements as described above. Down-regulation of FECH expression in the carcinoma cell lines corresponded to their potential to accumulate PpIX after ALA stimulation. Silencing of FECH expression by siRNA treatment For silencing of FECH mRNA expression by siRNA treatment, the colorectal carcinoma cell line LS174T was chosen. This cell line displayed strong FECH expression
on the mRNA and enzyme activity level, but showed only low PpIX fluorescence after ALA addition (Table 1). We used siRNA sequences directed toward three different regions of the coding region of human FECH , starting from base pairs 510, 1140, and 2107 of the FECH sequence (gene bank accession NM_001012515.1) in a concentration of 50 nM. In addition, a 50 nM mixture composed of all three sequences was used, and cells were treated with siRNA directed toward a sialyltransferase enzyme (EC 2.4.99.1) not related to heme biosynthesis. Most effective in FECH down-regulation was the siRNA sequence FECH-1140, which blocked 67% of the FECH expression found in mock-treated cells. After treatment of LS174T cells with siRNA FECH-1140, a strong PpIX fluorescence was observed (Fig. 3). Thus, siRNA treatment of LS174T cells led to a drastically increased PpIX fluorescence. PpIX accumulation after FECH siRNA treatment combined with ALA stimulation To study the effect of a combined treatment with FECH siRNA in conjunction with ALA, cells of the colorectal carcinoma line LS174T were treated twice with siRNA FECH-1140. Thereafter, ALA was added in a concentration of 0.1 mM ALA for 240 min. As a consequence, PpIX fluorescence was increased more than 50-fold by treatment with siRNA FECH-1140 compared to ALA treatment alone (Fig. 4). Images of treated LS174T cells were generated by two-photon microscopy (Figs. 5 and 6). Because of the high depth of field of two-photon microscopy, the images show sections through the LS174T cells where the fluorescence light collected stems from a layer of ⬃1 m thickness. Imaging was done 5 m above the coverslide surface. With cell diameters of ⬃10 m, the images shown in Figs. 5 and 6 represent sections through the axial center of the cells. The images show the fluorescence detected in the PpIX channel reduced by the fluorescence intensity detected in the autofluo-
Figure 2. FECH mRNA expression in different carcinomas of the gastrointestinal tract. FECH mRNA expression was determined by quantitative RT-PCR in different parts of the gastrointestinal tract. A panel of 18 cases with esophageal carcinomas, 20 cases with gastric carcinomas, 15 cases with colonic carcinomas, and 15 cases with rectal carcinomas, along with their non-malignant counterparts, was examined. Throughout the gastrointestinal tract, except in esophageal tissue, FECH expression was significantly higher in normal mucosa compared to carcinoma tissue (P⬍0.05; Wilcoxon test). Computational analysis of expression data was done using the ⌬⌬Ct method in comparison to ß-actin expression (see Materials and Methods). Abbreviations: CA, carcinoma tissue; NM, normal mucosa. x-axis: tissue samples; y-axis, relative amount of expression; *,°samples rated as extremes or outliers. 504
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TABLE 1. Comparison of FECH mRNA expression, FECH enzyme activity, and Pp IX fluorescence in an in vitro model of several human carcinoma cell lines
Cell line
HT29-P92 SW480 HT29-P37 MDA-MB-435 LS174T
FECH mRNA expression (relative amount)
FECH enzyme activity (nU/mg)
PpIX (nmol/106 cells)
0.526 ⫾ 0.03 0.551 ⫾ 0.02 0.696 ⫾ 0.02 0.921 ⫾ 0.08 1.000 ⫾ 0.05
8.92 ⫾ 0.6 10.92 ⫾ 4.1 12.33 ⫾ 3.8 18.33 ⫾ 1.9 23.54 ⫾ 6.3
16.96 ⫾ 4.1 27.94 ⫾ 3.8 19.15 ⫾ 3.2 0.73 ⫾ 0.3 1.76 ⫾ 0.3
Cells were cultured in 6-well plates for 5 days. Quantitative RT-PCR (Taqman) was used for determination of FECH mRNA expression. Computational analysis of expression data was done using the ⌬⌬Ct method with reference to ß-actin. Zn-Pp was used as a reaction product to determine FECH enzyme activity by HPLC-based fluorescence detection. Intracellular PpIX concentration was determined by time-resolved fluorescence spectroscopy using cells that were harvested after the addition of 0.1 mM aminolevulinic acid for 4 h (see Materials and Methods). As depicted in columns 2 and 3, FECH enzyme activity showed a strong correlation with mRNA expression (r2⫽0.95). Determination of PpIX shows that cell lines with high FECH enzyme activity displayed low PpIX fluorescence (r2⫽0.71). Given are the means of five independent measurements. Cells used were colorectal carcinoma cells HT29 (two different passages), SW480, and LS174T, and MDA-MB-435 cells, originally derived from a mammary carcinoma.
rescence channel. Since endogenous fluorescence emission takes place in a broad wavelength range, almost identical autofluorescence intensities are detected in both channels. Therefore, the distribution of the autofluorescence signal in the PpIX channel can be compensated for by subtracting the fluorescence intensities detected in the autofluorescence channel from the fluorescence signal detected in the PpIX channel. In addition, corresponding scatter plots show the correlation between the images detected at different de-
Figure 3. PpIX accumulation in cells treated with siRNA directed toward FECH mRNA. LS174T cells were treated two times (see Materials and Methods) with 50 nM siRNA directed toward different regions of the FECH mRNA sequence (FECH mRNA, transcript variant 1, gene bank accession NM_001012515.1). PpIX was quantitatively determined by time-resolved fluorescence spectroscopy. Abbreviations: mock, no siRNA treatment; other, siRNA toward sialyltransferase ST6GALI; FECH-510, siRNA directed toward the FECH sequence starting at bp 510; FECH-1140, siRNA directed toward the FECH sequence starting at bp 1140; FECH-2107, siRNA directed toward the FECH sequence starting at bp 2107; FECH-mixture, equal amounts of the siRNAs directed toward different regions of the FECH mRNA sequence. x-axis: siRNA treatment; y-axis: nmol PpIX per 106 LS174T cells. Given are the means of five independent measurements. PROTOPORPHYRIN-IX ACCUMULATION IN COLON CANCER
Figure 4. PpIX accumulation in cells treated with siRNA directed toward FECH mRNA. LS174T cells were treated two times (see Materials and Methods) with 50 nM siRNA directed toward different regions of the FECH mRNA sequence (FECH mRNA, transcript variant 1, gene bank accession NM_001012515.1). Thereafter, ALA was added in a concentration of 0.1 mM ALA for 4 h. PpIX was quantitatively determined by time-resolved fluorescence spectroscopy. PpIX fluorescence was increased almost 50-fold by treatment with siRNA FECH-1011 compared to ALA-treatment alone (mock). Abbreviations: mock, no siRNA treatment; other, siRNA toward sialyltransferase ST6GAL-I; FECH-510, siRNA directed toward the FECH sequence starting at bp 510; FECH-1140, siRNA directed toward the FECH sequence starting at bp 1140; FECH-2107, siRNA directed toward the FECH sequence starting at bp 2107; FECH mixture, equal amounts of the siRNAs directed toward different regions of the FECH mRNA sequence. x-axis: siRNA treatment; y-axis: nmol PpIX per 106 LS174T cells. Given are means of five independent measurements.
tection wavelengths, (i.e., PpIX at 635⫾20 nm and endogenous fluorescence at 535⫾17.5 nm). The use of scatter plots for signal decomposition has been demonstrated by Mo¨rtelmaier et al. (12). While mock-treated cells showed almost no fluorescence in the PpIX emission band (Fig. 5a, b), treatment with 50 nM FECH-1140 siRNA led to a strong increase in PpIX fluorescence as evidenced by the increased brightness of the image (Fig. 5c) and the occurrence of high fluorescence intensity spots apparent in the corresponding scatter plot up to values of 250 counts PpIX/ ms (Fig. 5d). Similarly, treatment with 1 mM ALA alone led to a strong increase of PpIX fluorescence compared with mock treatment (Fig. 6a, b). However, PpIX fluorescence intensity was much higher after combined treatment of LS174T cells with FECH1140 siRNA and ALA, yielding up to 2500 counts PpIX/ms (Fig. 6c, d).
DISCUSSION Hemes and heme proteins are vital components of virtually every cell. The last step of heme synthesis is incorporation of iron into PpIX, which takes place in the mitochondria catalyzed by the enzymeFECH. Selective PpIX-formation in human cancers, especially as a response to ALA medication, was suspected to be related to lowered FECH enzyme activity (5). We have determined FECH mRNA expression in gastrointestinal carcinomas using quantitative RT-PCR and were able to demonstrate a significant down-regulation of 505
Figure 5. Two-photon microscopy images of untreated LS174T cells (a) and cells treated with FECH-1140 siRNA (c). Images a and c show cells fluorescently labeled by PpIX. Fluorescence is as detected in the PpIX channel, corrected for the fluorescence intensity of the autofluorescence channel. Please note that the intensity scale in false colors has been cropped for better visibility, and intensity values that exceed the scale are shown in white. The sharp yellow spot in image a is an artifact. Corresponding scatter plots of two-photon microscopy images of LS174T cells (b) and cells treated with FECH-1140 siRNA (d) are presented as well. The scatter plots show the correlation pixel by pixel between images detected at different emission wavelengths. x-axis: endogenous autofluorescence at ⫽ 535 ⫾ 18 nm; y-axis: PpIX fluorescence at ⫽ 635 ⫾ 20 nm.
FECH mRNA expression in gastric, colonic, and rectal carcinomas (Figs. 1, 2). Investigation of an in vitro model using carcinoma cell lines different in PpIX formation showed that FECH mRNA expression correlated directly with FECH enzyme activity (Table 1). Furthermore, in this model, FECH down-regulation led to enhanced PpIX fluorescence, establishing a first evidence of causality. The final functional argument to prove the causal relationship between FECH expression/activity and PpIX formation is now provided by the fact that the observed ALA resistance in vitro can be overcome by siRNA interference directed toward FECH (Fig. 3). Sequence-specific posttranscriptional gene silencing by double-stranded RNA (dsRNA), also called 506
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RNA interference, is conserved in a diverse variety of organisms (13). RNA interference is mediated by siRNAs that are produced from long dsRNAs of exogenous or endogenous origin by an endonuclease of the ribonuclease-III type. The resulting siRNAs are ⬃21–23 nucleotides long and are incorporated into a nuclease complex. After binding to the RNA-induced silencing complex, complementary mRNA is cleaved (14). Here, we describe siRNA sequences effective in reducing FECH expression by 67% on transient transfection, leading to a dramatic PpIX accumulation in vitro in an otherwise PpIX-negative cell line (Fig. 3). Under ALA substitution, siRNA transfection led to an increase in the accumulated PpIX by a factor of 50 (Fig. 4),
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Figure 6. Two-photon microscopy images of LS174T cells treated with ALA (a) and cells treated with FECH-1140 siRNA in conjunction with ALA (c). Images a and c show cells fluorescently labeled by PpIX are displayed. Fluorescence is as detected in the PpIX channel, corrected for the fluorescence intensity of the autofluorescence channel. Please note that the intensity scale in false colors has been cropped for better visibility, and intensity values that exceed the scale are shown in white. Corresponding scatter plots of two-photon microscopy images of LS174T cells treated for 4 h with 1 mM ALA alone (b) and cells treated with FECH-1140 siRNA in conjunction with 1 mM ALA (d) are presented as well. The scatter plots show the correlation pixel by pixel between images detected at different emission wavelengths. x-axis: endogenous autofluorescence at ⫽ 535 ⫾ 18 nm; y-axis: PpIX fluorescence at ⫽ 635 ⫾ 20 nm.
resulting in strong cytoplasmatic red fluorescence (Figs. 5, 6). Thus, silencing of FECH expression by siRNA treatment led to strongly increased PpIX fluorescence. This series of experiments establishes for the first time a strong causal relationship between FECH expression and PpIX formation with or without ALA substitution. The logical step forward from this observation is to ask how and why this apparently rather constant phenomenon occurs and, moreover, whether this knowledge can be exploited for cancer diagnosis or treatment. FECH is found in its mature homodimeric form bound to the matrix side of the inner mitochondrial PROTOPORPHYRIN-IX ACCUMULATION IN COLON CANCER
membrane. Translocation into the mitochondrion is energy dependent and involves proteolysis to remove the leader sequence and assembly of the (2Fe-2S) cluster (15). Results of Taketani et al. (16) indicated that intracellular iron levels, via the iron-sulfur cluster center at the C-terminus, regulate the protein expression of mammalian FECH and that this contributes to the regulation of the heme biosynthesis. Treatment of erythroleukemia cells with an iron chelator resulted in a decreased FECH protein level and heme content. However, since in these cells the translation of ALA-S, the first enzyme of heme synthesis, is also seen to be repressed by the loss of intracellular iron, no increased accumulation of PpIX was found under these condi507
tions. The effect of mRNA expression on the regulation of FECH activity is not fully understood. Magness et al. (17), who studied FECH expression during hematopoietic development of embryonic stem cells, speculated on a translational control mechanism involving repressive stabilization of FECH mRNA. Interestingly, exposure of several cell lines to hypoxia for 18 h resulted in a significant increase in FECH mRNA expression (18). In the in vitro model studied here, we found a good correlation between FECH mRNA expression and enzyme activity. Down-regulation of FECH in the cancer tissues could be due to promoter methylation and remains the subject of further investigations. On the other hand, FECH siRNA silencing might be a versatile tool for molecular imaging (19) and cancer treatment. Currently, molecular optical imaging relies on transfection with gene sequences encoding fluorescent proteins (20) or on exogenously administered specifically interfering fluorescing probes including fluorescent beacons activated by tumor-associated proteases (21). We report here, for the first time, the functional use of siRNA for cellular imaging. This approach has several advantages. Sequences encoding fluorescent probes have to cross cellular and nuclear membranes before being transcribed, while siRNA based compounds find their target directly within the cytoplasm. The presented approach exhibits no relevant toxicity because siRNA silencing of FECH led to an endogenous and nontoxic fluorescence by affecting the cellular heme metabolism. Moreover, because of the omnipresence of the heme-synthesis pathway, targeted application of siRNA may provide a general means for cellular imaging. The applicability of all approaches in vivo is hindered by insufficient delivery into the cell. Most likely, a selectivity mechanism that directs siRNA constructs specifically to tumor cells is needed. Although direct systemic application of siRNA recently has been used successfully (22), rapid degradation might hamper its efficiency. An alternative might be local application of naked siRNA using a gene gun approach (23) or siRNA delivery using receptor-mediated endocytosis through liposomes or other carriers (24, 25). For achieving successful and highly selective siRNA in vivo delivery, we are planning to deliver the siRNAs to tumor tissues carried in cationic liposomes endowed with a ligand which binds to its receptors on the tumor cell membrane. Transferrin coupled to the PEG terminus has been shown to provide a good accessibility to its ligand receptor molecules and trigger receptor-mediated internalization of the entire siRNA-carrying liposome package into the cell interior (24). Clinical applications range from a better definition of surgical margins to a better detection of flat precancerous lesions and early tumors (26, 27). Moreover, endogenous formation of PpIX resultant from local application of FECH-siRNA, alone or in conjunction with 5-ALA, might increase sensitivity and capability of endoscopic diagnosis. FECH silencing also might prove suitable for therapeutic approaches because, via laser light treatment, 508
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selective destruction of siRNA-transfected cells can be achieved easily. With regard to conventional cytotoxic constructs used in gene therapy approaches, the cytotoxic effect in these cases requires and relies on a local component, light activation. Therefore, if a gene therapeutic construct has affected cells in light-protected areas of the body, a restriction of specific light activation to the target areas may provide cytotoxicity with limited side effects. In conclusion, using siRNA technology we demonstrated that down-regulation of FECH mRNA expression is a major cause of PpIX fluorescence in carcinoma tissues. This approach may open a whole range of imaging applications, from basic research to clinical applications. We thank Jan Voigt and Sabine Grigull for their excellent technical assistance. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Mo534/4).
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Received for publication May 4, 2007. Accepted for publication August 16, 2007.
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