Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937
Takashi AmoNoriaki KawanishiMasataka UchidaHirofumi FujitaEri OyanagiToshihiko UtsumiTetsuya OginoKeiji InoueTaro ShuinKozo UtsumiJunichi Sasaki
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Abstract Photodynamic therapy (PDT) for tumors is based on the tumor‐selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5‐aminolevulinic acid (ALA)‐based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl‐xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase‐3 activation, phosphatidylserine (PS) externalization. PDT‐induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright © 2009 John Wiley & Sons, Ltd.Keywords:
Ferrochelatase
U937 cell
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Ferrochelatase
Erythropoietic protoporphyria
Protoporphyrin IX
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Ferrochelatase
Erythropoietic protoporphyria
Protoporphyrin IX
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Ferrochelatase
Erythropoietic protoporphyria
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Ferrochelatase
Erythropoietic protoporphyria
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Ferrochelatase
Protoporphyrin IX
Tetrapyrrole
Enzyme Kinetics
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Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron and protoporphyrin to form heme. The activity of ferrochelatase is deficient in the inherited disease protoporphyria. In this study, murine ferrochelatase cDNAs were obtained by screening cDNA libraries with an oligonucleotide probe. The derived amino acid sequence of murine ferrochelatase has 47% identity with the recently cloned Saccharomyces cerevisiae ferrochelatase, but it is not significantly similar to other published sequences. Results of Southern blotting are consistent with a single murine ferrochelatase gene, while Northern blotting demonstrates two ferrochelatase transcripts in all tissues examined. The ferrochelatase protein and mRNAs have different relative concentrations in different tissues. The cloning of murine ferrochelatase cDNAs provides the basis for future studies on ferrochelatase gene expression and on the identification of the molecular defect in protoporphyria.
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Erythropoietic protoporphyria
Protoporphyrin IX
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Abstract Ferrochelatase deficiency has been shown in both porphyria variegata (PV) and erythropoietic protoporphyria (EPP). It has been suggested that in PV there is a decrease in the enzyme, whereas in EPP the enzyme is unstable. In the present study ferrochelatase activity was measured in skin fibroblasts from three patients with PV and three normal subjects. The enzymatic activity in the patients with PV (17.5 ± 4.5 pmoles heme formed per 10 7 fibroblasts per hour) was 50% of that of the control group (31.0 ± 3.2 pmoles heme formed per 10 7 fibroblasts per hour). This supports the contention that the enzyme is deficient in PV and that an inactive ferrochelatase is the primary deficiency in this type of porphyria.
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Ferrochelatase, which catalvses the last stop in heme biosynthesis, i. e. the insertion of Fe+ + into protoporphyrin Ⅸ, present in all celis, particularly aboundanty in erythroid cells during hemoglobiniza-tion. In present study, we observed expression of ferrochelatase mRNA in different statuses of intracellular iron and heme synthesis in non- induced and DMSO induced murine erythroleukemia (MEL) celis with 1. 5 kb cDNA as probe. The results were as follows: ① In non - induced celis the expression of ferrochelatase mRNA had no changes. ② The expression of ferrochelatase mRNA had increased in induced celis. ③ The level of intracellular iron in both non - induced and induced cells has effect on ex-pression of ferrochelatase mRNA bt faintful: when iron in excess, the expression of mRNA was increased; when iron in deficiency, the expression was decreased. ④ The inhibition of heme synthesis by INH decreases the expression of ferrochelatase mRNA in induced cells.
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The ferrochelatase-inhibitory activity, porphyrin-inducing activity, and cytochrome P-450- and heme-destructive effects of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were studied in chick embryo liver cells. The ferrochelatase-inhibitory activity of the 4-butyl, 4-pentyl, 4-hexyl, and 4-cyclopropylmethyl analogues of DDC was considered to be due to the formation of the corresponding N-alkylporphyrins. These N-alkylporphyrins were isolated from the livers of phenobarbital-pretreated rats following administration of the corresponding DDC analogues. The 4-isobutyl analogue did not have ferrochelatase-inhibitory activity despite its ability to cause formation of an N-isobutylporphyrin in rat liver. The 4-chloromethyl analogue of DDC inhibited ferrochelatase activity. The inability to isolate an N-alkylporphyrin from rat liver with this analogue may be due to its lability. The porphyrin-inducing activity of these analogues depended on their ferrochelatase-inhibitory potency and lipophilicity. The DDC analogues caused cytochrome P-450 and heme destruction. The relative ferrochelatase-inhibitory activity of the DDC analogues has implications for a postulated model of the binding of porphyrins in the ferrochelatase active site.
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Ferrochelatase (heme synthase, protoheme ferrolyase [EC 4.99.1.1]), the final enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous ion into protoporphyrin IX to produce protoheme IX. The thorough understanding of the enzyme is prerequisite to elucidating the regulation of iron and heme metabolism. The enzyme's activity is found on the inner mitochondrial membrane of a variety of mammalian cells. The enzyme catalyzes the chelation not only of iron but also of divalent metal ions including cobalt and zinc, and the activity is affected by various metals and lipids. The molecular weights of eukaryotic ferrochelatases are 40,000-42,000 daltons. Complementary DNA (cDNA) encoding ferrochelatase from mouse, human, yeast and bacteria have been isolated, and the derived amino acid sequences show 27-88% homologies among species. The expression of ferrochelatase seems to occur in all living cells, and to play an important role in the regulation of heme biosynthesis. Ferrochelatase is markedly induced at the transcriptional level during erythroid differentiation when iron uptake by cells and hemoglobin synthesis are upregulated. This induction can be explained by the existence of sequences characteristic of erythroid-related genes. The gene has been mapped to human chromosome 18q21.3 and contains 11 exons with a size of about 45 kilobases. Once the gene for human ferrochelatase is cloned, the molecular basis and clinical diagnosis of erythropoietic protoporphyria, caused by a deficiency of ferrochelatase, will become possible. This review summarizes recent advances in ferrochelatase research and suggests important subjects for future research.
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