Showing papers on "Protoporphyrin IX published in 2007"

Inhibition of heme oxygenase-1 by zinc protoporphyrin IX reduces tumor growth of LL/2 lung cancer in C57BL mice.

Abstract: Heme oxygenase (HO)-1 is a key player reducing cytotoxicity and enhancing protumoral effects of nitric oxide (NO). We examined zinc protoporphyrin (ZnPP) IX, an HO-1 inhibitor, to affect tumor growth of LL/2 mouse lung cancer cells. ZnPPIX reduced HO-1 expression and HO activity in LL/2 cells, whereas cobalt PPIX (CoPPIX), an HO-1 activator, increased both. LL/2 cells treated with sodium nitropurusside, an NO donor, showed growth inhibition dose-dependently, which was enhanced by ZnPPIX cotreatment, but was reduced by CoPPIX. In mice tumors, ZnPPIX decreased HO-1 expression. LL/2-tumors were found in 88% (7/8) vehicle-treated mice, whereas tumors were found in 38% (3/8) and 25% (2/8) mice treated with 5 and 20 μg/mouse ZnPPIX, respectively (p = 0.0302). Tumor growth was inhibited dose-dependently by ZnPPIX. Vascular endothealial growth factor concentration in tumors was reduced by ZnPPIX (p = 0.0341). Microvessel density (MVD) in ZnPPIX-treated tumors was lower than that in vehicle-treated tumors (p = 0.0362). Apoptotic cell count in ZnPPIX-treated tumors was higher than that in vehicle-treated tumors (p = 0.0003). In contrast, CoPPIX treatment increased HO-1 expression, enhanced tumorigenicity and MVD and reduced apoptosis. From these findings, inhibition of HO-1 by ZnPPIX provides relevant antitumoral effects. © 2006 Wiley-Liss, Inc.

The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana

Abstract: The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress.

Liver disease in erythropoietic protoporphyria: insights and implications for management

Abstract: The porphyrias are a group of disorders caused by defects in haem biosynthesis (fig 1). Of the seven main types of porphyria recognised, two are characterised by associated liver disease (table 1). In porphyria cutanea tarda it is the liver disease which leads to the onset of the porphyria, characterised by blistering, hirsutes and skin fragility of sun-exposed skin. A number of different liver diseases may precipitate porphyria cutanea tarda including haemochromatosis, alcoholic liver disease and hepatitis C. In contrast, in erythropoietic protoporphyria (EPP) it is the porphyria itself which leads to liver disease, due to progressive deposition and accumulation of insoluble protoporphyrin IX in hepatocytes and bile canaliculi. View this table: Table 1 Liver disease and the porphyrias: names and patterns of inheritance for the seven main clinical variants of porphyria, highlighting those characterised by concomitant liver disease Figure 1 The haem biosynthetic pathway showing the enzyme deficiency associated with porphyria cutanea tarda (PCT) and erythropoietic protoporphyria (EPP). The final step in this pathway involves the incorporation of iron into the middle of the ring structure of protoporphyrin IX to form haem. EPP is an inborn error of haem biosynthesis caused by mutations in the gene encoding the mitochondrial enzyme ferrochelatase (FECH), the final enzyme in the haem biosynthetic pathway (fig 1).1–5 It was first described by Magnus et al in 1962.6 Ferrochelatase catalyses the insertion of ferrous iron into protoporphyrin to form haem, and when defective or deficient, accumulation of protoporphyrin ensues. Ferrochelatase is active in cells that produce haem including erythroid precursors in the bone marrow7 and hepatocytes.8 However, the majority of protoporphyrin (80% or more) originates from bone marrow with most of the remainder generated by the liver (fig 2).7,9 Figure 2 The fate of protoporphyrin IX in erythropoietic protoporphyria. Protoporphyrin accumulates in the maturing red blood cells …

Parasiticidal effect of δ‐aminolevulinic acid‐based photodynamic therapy for cutaneous leishmaniasis is indirect and mediated through the killing of the host cells

Abstract: Several clinical reports have shown promising, but not optimal, results from photodynamic therapy with delta-aminolevulinic acid-derived protoporphyrin IX, termed ALA-PDT, as a treatment for cutaneous leishmaniasis (CL). Therefore, understanding the basis of the phototoxic response of Leishmania parasites to ALA-PDT may be critical for optimization. We report here both in vitro and in vivo mechanistic studies of ALA-PDT against CL. Following in vitro co-incubation of Leishmania major with 0.1 microM ALA, the PpIX concentration remained at the basal level, whereas after co-incubation with 0.1 microM exogenous PpIX, the PpIX level was 100-fold higher. No differences in ALA-derived PpIX levels were detected between Leishmania-infected and non-infected J774.2 cells, and PDT did not demonstrate any parasiticidal effects on amastigotes. In contrast, in vivo topical ALA-PDT, performed on a murine CL model, resulted in significant reductions of the parasite loads and vigorous tissue destruction. After ALA-PDT, a dramatically decreased percentage of macrophages and increased levels of interleukin-6 were observed in the infected skin. The clinical outcome observed with ALA-PDT is likely the result of unspecific tissue destruction accompanied by depopulation of macrophages rather than direct killing of parasites.

Quantitative spectroscopic analysis of 5-aminolevulinic acid-induced protoporphyrin IX fluorescence intensity in diffusely infiltrating astrocytomas.

Abstract: The fluorescence of protoporphyrin IX (PpIX) induced endogenously by 5-aminolevulinic acid (5-ALA) administration has recently been used for the intraoperative visualization of glioma tissues. To increase the sensitivity of photodetection, the emission spectra of 5-ALA-induced PpIX fluorescence was quantitatively measured in tissues taken from six cases of en bloc resected diffusely infiltrating astrocytomas (2 diffuse astrocytomas, 2 anaplastic astrocytomas, and 2 glioblastomas), and the correlation assessed between the fluorescence intensity and histological features. A total of 65 slices were analyzed by ex vivo spectroscopy. The ratio of the peak emission intensity to reflected excitation intensity or fluorescence intensity ratio was less than 0.001 for all 36 non-tumor tissues. The tissues with glioblastoma morphology had a fluorescence intensity ratio in excess of 0.090. The spectroscopic fluorescence intensity was positively correlated with the MIB-1 labeling index as an indicator of proliferation activity, the CD31-microvessel density as a pan-endothelial marker, and the vascular endothelial growth factor expression as an angiogenetic factor. The MIB-1 proliferation index was the most powerful determinant, suggesting that higher cell proliferation may govern preferential PpIX accumulation in glioma cells. This preliminary study suggests that spectroscopic analysis may be useful for optimizing the removal of diffuse gliomas.

Macromolecular delivery of 5-aminolaevulinic acid for photodynamic therapy using dendrimer conjugates

Abstract: Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (5-ALA) has been widely used in photodynamic therapy. However, cellular uptake of 5-ALA is limited by its hydrophilicity, and improved means of delivery are therefore being sought. Highly branched polymeric drug carriers known as dendrimers present a promising new approach to drug delivery because they have a well-defined structure capable of incorporating a high drug payload. In this work, a dendrimer conjugate was investigated, which incorporated 18 aminolaevulinic acid residues attached via ester linkages to a multipodent aromatic core. The ability of the dendrimer to deliver and release 5-ALA intracellularly for metabolism to the photosensitizer, protoporphyrin IX, was studied in the transformed PAM 212 murine keratinocyte and A431 human epidermoid carcinoma cell lines. Up to an optimum concentration of 0.1 mmol/L, the dendrimer was significantly more efficient compared with 5-ALA for porphyrin synthesis. The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure, together with minimal dark toxicity. Cellular uptake of the dendrimer occurs through endocytic routes predominantly via a macropinocytosis pathway. In conclusion, macromolecular dendritic derivatives are capable of delivering 5-ALA efficiently to cells for sustained porphyrin synthesis. [Mol Cancer Ther 2007;6(3):876–85]

Fluorescence-guided resection of metastatic brain tumors using a 5-aminolevulinic acid-induced protoporphyrin IX: pathological study

Abstract: We performed a pathological study to identify the locus of production of protoporphyrin IX (PPIX) in human metastatic brain tumors. Patients with metastatic brain tumors (n = 11) received 1 g of 5-aminolevulinic acid (5-ALA) perorally 2 h before undergoing surgery. The target region was exposed to laser light with a peak wavelength of 405 ± 1 nm and an output of 40 mW. Tissue samples from the tumor bulk and surrounding areas were examined by histological and fluorescence methods. Of the 11 tumors, 9 manifested PPIX fluorescence in the tumor bulk and peritumoral brain tissue. Our findings indicate that PPIX fluorescence can be observed in peritumoral edematous areas that are free of neoplastic cells, because PPIX produced by neoplastic cells leaks into the surrounding edematous area.

Substrate interactions with human ferrochelatase

Abstract: Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Human ferrochelatase is a homodimeric, inner mitochondrial membrane-associated enzyme that possesses an essential [2Fe-2S] cluster. In this work, we report the crystal structure of human ferrochelatase with the substrate protoporphyrin IX bound as well as a higher resolution structure of the R115L variant without bound substrate. The data presented reveal that the porphyrin substrate is bound deep within an enclosed pocket. When compared with the location of N-methylmesoporphyrin in the Bacillus subtilis ferrochelatase, the porphyrin is rotated by ≈100° and is buried an additional 4.5 A deeper within the active site. The propionate groups of the substrate do not protrude into solvent and are bound in a manner similar to what has been observed in uroporphyrinogen decarboxylase. Furthermore, in the substrate-bound form, the jaws of the active site mouth are closed so that the porphyrin substrate is completely engulfed in the pocket. These data provide insights that will aid in the determination of the mechanism for ferrochelatase.

Inhibition of heme oxygenase-1 increases responsiveness of melanoma cells to ALA-based photodynamic therapy.

Abstract: Based on the observation that 5-aminolevulinic acid (ALA) induces the expression of heme oxygenase-1 (HO-1) in cultured melanoma cells, the role of HO-1 on the effectiveness of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) was examined. Transcriptional activation of the HO-1 gene is considered to be an adaptive response to oxidative and cellular stress and confers a protective capacity against cell and tissue injury, which could affect the responsiveness to ALA-PDT. A time-dependent accumulation (0-16 h) of protoporphyrin IX (PPIX) within melanoma cells was seen after incubation with ALA (0.5 mM ALA). Over the same time interval, a significant increase (up to 25-fold) in HO-1 protein expression was observed. Thus, the production and degradation of PPIX (via heme by HO-1) were simultaneously enhanced, leading to a reduced intracellular concentration of the photodynamically active substance PPIX. Diminishing HO-1 activity by the HO-1 inhibitor tin protoporphyrin IX (SnPPIX) significantly enhanced the formation of PPIX up to 1.8 fold. A further strong increase in HO-1 protein expression (up to 128-fold) was seen after ALA-PDT treatment. Induction of HO-1 is an essential step in the 'rescue response' of tumor cells. The pharmacological inhibition of HO-1 activity by SnPPIX leads to a considerable increase in the sensitivity of tumor cells to ALA-PDT treatment. At low radiation doses (0.42 J/cm(2)), the percentages of death cells increased significantly from 7.3+/-1.3% to 43.7+/-6.4%. This effect could be further intensified by cellular depletion of HO-1 mRNA by siRNA. The combination of pharmacological inactivation of HO-1 with gene silencing led to an increase in the death rate of up to 54.1+/-8.6%. The results presented indicate that HO-1 can play a protective role against ALA-PDT mediated cytotoxicity so that a specific inhibition of HO-1 activity and/or expression might be used to increase the efficacy of ALA-based photodynamic therapy.

Bactericidal effect of photodynamic therapy against methicillin-resistant Staphylococcus aureus strain with the use of various porphyrin photosensitizers.

Abstract: Photodynamic therapy (PDT) is based on photosensitizers activated by light of appropriate wavelength. Their activation leads to generation of singlet oxygen and free radicals responsible for the cytotoxic effect. The aim of this project was to compare the bactericidal effect of PDT using different porphyrin photosensitizers against a methicillin-resistant Staphylococcus aureus strain. Exogenous sensitizers (protoporphyrin IX and newly synthesized derivative, protoporphyrin diarginate) induced a 3 log10-unit reduction in bacterial viable counts. With the use of endogenous, ALA-induced porphyrins, a 1.6 log10-unit reduction was obtained. The sensitizers tested executed their antibacterial activity with no essential change in the antibiotic resistance pattern of the studied strain.

Comparison between sonodynamic effect with protoporphyrin IX and hematoporphyrin on sarcoma 180.

Abstract: Purpose The comparison between sonodynamic antitumor effect with protoporphyrin IX (PPIX) and hematoporphyrin (Hp) at a concentration of 5 mg/kg on Sarcoma 180 (S180) cells was studied in vivo, and the potential cell damage mechanism was also investigated.

Study of the efficacy and mechanism of ALA‐mediated photodynamic therapy on human hepatocellular carcinoma cell

Abstract: Objectives: To examine the efficacy and mechanism of δ- or 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on a human hepatocellular carcinoma cell line. Materials and methods: The optimal uptake of photosensitizer ALA in HepG2 (p53 wild) cells was investigated by means of spectrometric measurement. Cell viability was determined by trypan blue exclusion assay. Morphological apoptotic changes in HepG2 cells before and after ALA-mediated PDT were determined by microscopic examination. Detection of apoptotic bodies was examined by DAPI staining. The changes in p53 expression were revealed by the immunostaining method. Results: ALA/protoporphyrin IX (PpIX) was mainly located in the cytoplasm of HepG2 cells. The maximal cellular uptake occurred after 18 h in vitro incubation. The photocytotoxic assay showed that ALA PDT induced 80% killing at 2 mM drug dose and 2 J/cm2 light intensity. Up to 70% of cells showed membrane blebbing and positive DAPI staining, indicating that ALA–PDT-mediated cell death was predominantly via apoptosis. In addition, p53 was upregulated after treatment, implying that p53 might evoke apoptotic cell death. Conclusions: HepG2 cell line is sensitive to ALA-mediated PDT. ALA–PDT induces apoptosis in the HepG2 cell line that may be mediated by a p53-dependent pathway.

Endoscopic identification and biopsy sampling of an intraventricular malignant glioma using a 5-aminolevulinic acid-induced protoporphyrin IX fluorescence imaging system. Technical note.

Abstract: ✓Several neurosurgical studies have provided descriptions of the utility of fluorescence-guided tumor resection using a microscope. However, fluorescence-guided endoscopic detection of a deep-seated brain tumor has not yet been reported. The authors report their experience with an endoscopic biopsy procedure for a malignant glioma within the third ventricle using a 5–aminolevulinic acid (5-ALA)–induced protoporphyrin IX fluorescence imaging system. A 5-ALA–induced fluorescence image of an intraventricular tumor is barely visible with the typical fluorescence endoscopic system used in other clinical fields because the intensity of excitation light at wavelengths of 390 to 405 nm through a cutoff filter is too weak to delineate a brain tumor. The technique described in this study made use of a laser illumination system with a high-powered output that delivered a violet-blue light at wavelengths of 405 nm. In addition, a common ultraviolet cutoff filter was fitted between the endoscope and the high-sensitivi...

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

Abstract: PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent Km value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 μM, with a Vmax of 4.27 μM·min−1·mg−1 and a catalytic activity kcat of 6.0 s−1. Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe392 in substrate ring A stacking. Mutations of Leu356, Leu372 and Arg98 increased kcat values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased Km values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure–function relationship underlying detrimental human enzyme defects.

Amino acid residues His183 and Glu264 in Bacillus subtilis ferrochelatase direct and facilitate the insertion of metal ion into protoporphyrin IX.

Abstract: Ferrochelatase catalyzes the terminal step in the heme biosynthetic pathway, i.e., the incorporation of Fe(II) into protoporphyrin IX. Various biochemical and biophysical methods have been used to probe the enzyme for metal binding residues and the location of the active site. However, the location of the metal binding site and the path of the metal into the porphyrin are still disputed. Using site-directed mutagenesis on Bacillus subtilis ferrochelatase we demonstrate that exchange of the conserved residues His183 and Glu264 affects the metal affinity of the enzyme. We also present the first X-ray crystal structure of ferrochelatase with iron. Only a single iron was found in the active site, coordinated in a square pyramidal fashion by two amino acid residues, His183 and Glu264, and three water molecules. This iron was not present in the structure of a His183Ala modified ferrochelatase. The results strongly suggest that the insertion of a metal ion into protoporphyrin IX by ferrochelatase occurs from a metal binding site represented by His183 and Glu264.

Relation between intracellular location and photodynamic efficacy of 5-aminolevulinic acid-induced protoporphyrin IX in vitro. Comparison between human glioblastoma cells and other cancer cell lines.

Abstract: A promising clinical application of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PP IX) is fluorescence detection and photodynamic treatment of residual tumour tissue during surgical resection of high grade malignant glioma. U373 MG human glioblastoma cells were used as a model system to study the relation between intracellular location and photodynamic efficacy of 5-ALA-induced PP IX in more detail. Therefore, ultra-sensitive fluorescence microscopy, using either optical excitation of whole cells or selective excitation of the plasma membrane by an evanescent electromagnetic field, was combined with quantitative measurements of intracellular porphyrin amount and phototoxicity. Glioblastoma cells accumulated PP IX to a moderate extent as compared to T47D breast cancer cells (high accumulation) or OV2774 ovarian cancer cells (low accumulation). Although photodynamic inactivation of the different cell lines (decreasing in the order T47D > U373 MG > OV2774) seemed to be directly related to PP IX accumulation, examination of the data in more detail revealed that photodynamic efficacy per photosensitizer molecule (PE) was about two times higher in glioblastoma and ovarian cancer cells as compared to breast cancer cells. The different photodynamic efficacy of PP IX was related to the different intracellular location. In contrast to breast cancer cells where PP IXfluorescence was localized in small granules, PP IXfluorescence in glioblastoma cells and ovarian cancer cells originated mainly from cellular membranes. Thus, the intracellular location of PP IX in a predominantly lipophilic environment, characterized by a comparably high photostability (probed by photobleaching and photoproduct formation) and a lower degree of porphyrin aggregation (probed previously by fluorescence decay kinetics), seems to be the key factor for high photodynamic efficacy of 5-ALA-induced PP IX. In the case of OV2774 ovarian cancer cells, however, a low PP IX accumulation limited cell inactivation upon irradiation, whereas the results obtained for glioblastoma cells are encouraging to develop PDT to an additional therapeutic option for the treatment of tumour margins in patients who underwent fluorescence-guided resection of high grade malignant glioma after 5-ALA administration.

Direct measurement of metal ion chelation in the active site of human ferrochelatase.

Abstract: The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (EC 4.99.1.1). We demonstrate that pre-steady state human ferrochelatase (R115L) exhibits a stoichiometric burst of product formation and substrate consumption, consistent with a rate-determining step following metal ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k approximately 1 s-1) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady state data reveals that the rate-determining step in the overall reaction, conversion of free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal ion chelation, with a rate constant of 0.96 s-1, is approximately 10 times faster than the rate-determining step in the steady state (kcat = 0.1 s-1). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a 7-fold decrease in kcat and a 3-fold decrease in kchel. This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches.

5-Aminolevulinic acid-induced protoporphyrin-IX accumulation and associated phototoxicity in macrophages and oral cancer cell lines.

Abstract: Studies were carried out on 5-aminolevulinic acid (ALA)-induced protoporphyrin (PpIX) synthesis in mice peritoneal macrophages and two human oral squamous cell carcinoma (OSCC) cell lines NT8e and 4451. Cells were treated with 200 microg/ml ALA for 15 h and PpIX accumulation was monitored by spectrofluorometry and phototoxicity to red light (630+/-20 nm) was measured by MTT assay. PpIX accumulation was higher in macrophages as compared to OSCC cells under both normal serum concentration (10%) and conditions of serum depletion. The results on phototoxicity measurements correlated well with the levels of PpIX accumulation in both macrophages and cancer cells. While red light caused 20% phototoxicity in macrophages, no phototoxicity was seen in 4451 cells at 10% serum. Decrease in serum concentration to 5% and 1% led to higher phototoxicity corresponding to 40% and 70% in macrophages and 10% and 15% in 4451 cells. Similar results were obtained in NT8e cell line. Propidium iodide staining followed by fluorescence microscopic observations on photodynamically treated co-culture of murine or human macrophages and cancer cells showed selective damage to macrophages. These results suggest that in OSCC, macrophages would contribute more to tumor PpIX level than tumor cells themselves and PDT may lead to selective killing of macrophages at the site of treatment. Since macrophages are responsible for production and secretion of various tumor growth mediators, the effect of selective macrophage killing on the outcome of PDT would be significant.

Direct Comparison of δ-Aminolevulinic Acid and Methyl-Aminolevulinate-Derived Protoporphyrin IX Accumulations Potentiated by Desferrioxamine or the Novel Hydroxypyridinone Iron Chelator CP94 in Cultured Human Cells

Abstract: Aminolevulinic acid photodynamic therapy (ALA-PDT) is a cancer therapy that combines the selective accumulation of a photosensitizer in tumor tissue with visible light (and tissue oxygen) to produce reactive oxygen species. This results in cellular damage and ablation of tumor tissue. The use of iron chelators in combination with ALA has the potential to increase the accumulation of the photosensitizer protoporphyrin IX (PpIX) by reducing its bioconversion to heme. This study compares directly for the first time the effects of the novel hydroxypyridinone iron chelating agent CP94 and the more clinically established iron chelator desferrioxamine (DFO) on the enhancement of ALA and methyl-aminolevulinate (MAL)-induced PpIX accumulations in cultured human cells. Cultured human cells were incubated with a combination of ALA, MAL, CP94 and DFO concentrations; the resulting PpIX accumulations being quantified fluorometrically. The use of iron chelators in combination with ALA or MAL was shown to significantly increase the amount of PpIX accumulating in the fetal lung fibroblasts and epidermal carcinoma cells; while minimal enhancement was observed in the normal skin cells investigated (fibroblasts and keratinocytes). Where enhancement was observed CP94 was shown to be significantly superior to DFO in the enhancement of PpIX accumulation.

Breast Cancer Resistance Protein (Bcrp1/Abcg2) is expressed in the harderian gland and mediates transport of conjugated protoporphyrin IX

Abstract: Proper regulation of intracellular levels of protoporphyrin IX (PPIX), the direct precursor of heme, is important for cell survival A deficiency in ferrochelatase, which mediates the final step in heme biosynthesis, leads to erythropoietic protoporphyria (EPP), a photosensitivity syndrome caused by the accumulation of PPIX in the skin We have previously shown that mice with a deficiency in the ABC transporter Bcrp1/Abcg2 display a novel type of protoporphyria This protoporphyria is mild compared with ferrochelatase-dependent EPP, and in itself not sufficient to cause phototoxicity, but it might exacerbate the consequences of other porphyrias In this study, we identified the mouse harderian gland as a novel expression site of Bcrp1 Because of its pronounced role in porphyrin secretion, the harderian gland presents a useful tool to study the mechanism of Bcrp1-related protoporphyria and transport of porphyrins Bcrp1(-/-) harderian gland displayed a highly increased accumulation of PPIX glycoconjugates, and a similar shift was seen in Bcrp1(-/-) liver Tear- and hepatobiliary excretion data suggest that Bcrp1 controls intracellular levels of PPIX by mediating high affinity transport of its glycoconjugates and possibly low-affinity transport of unconjugated PPIX This mechanism may allow cells to prevent or reduce cytotoxicity of PPIX under excess conditions, without spillage under physiological conditions where PPIX is needed