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Synthesis of protoporphyrin IX (PpIX) by malignant cells is essential for the success of ALA-based photodynamic therapy (PDT). Two key enzymes that were described as affecting PpIX accumulation during ALA treatment are porphobilinogen deaminase (PBGD) and ferrochelatase. Here, we show that down regulation of ALA dehydratase (ALAD) expression and activity by specific shRNA induced a marked decrease in PpIX synthesis in K562 erythroleukemic cells. Photo-inactivation efficacy following ALA-PDT was directly correlated with ALAD-silencing and cellular levels of PpIX. MTT metabolism following ALA-PDT was shown to be 60% higher in ALAD-silenced cells in comparison to control cells, indicating that mitochondria were protected in the silenced cells. Morphological analysis by scanning electron microscopy (SEM) of cells treated by ALA-PDT showed no morphological changes in ALAD-silenced cells, in contrast to controls exhibiting cell deformations and lysis. Membrane integrity following ALA-PDT was kept intact and undamaged in ALAD-silenced cells as examined by Annexin V-FITC/PI staining and LDH-L leakage. We conclude that ALAD, although it is present in the cell at abundant levels, has a major and limiting role in regulating PpIX synthesis and ALA-PDT outcome.
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G. Berkovitch, D. Doron, A. Nudelman, Z. Malik and A. Rephaeli, Novel multifunctional acyloxyalkyl ester prodrugs of 5-aminolevulinic acid display improved anticancer activity independent and dependent on photoactivation, J. Med. Chem., 2008, 51, 7356–7369.
M. J. Gerritsen, T. Smits, M. M. Kleinpenning, P. C. van de Kerkhof, P. E. van Erp, Pretreatment to enhance protoporphyrin IX accumulation in photodynamic therapy, Dermatology, 2009, 218, 193–202.
S. L. Gibson, J. J. Havens, L. Metz and R. Hilf, Isδaminolevulinic acid dehydratase rate limiting in heme biosynthesis following exposure of cells toδaminolevulinic acid?, Photochem. Photobiol., 2001, 73, 312–317.
L. Greenbaum, Y. Gozlan, D. Schwartz, D. J. Katcoff and Z. Malik, Nuclear distribution of porphobilinogen deaminase (PBGD) in glioma cells: a regulatory role in cancer transformation?, Br. J. Cancer, 2002, 86, 1006–1011.
B. Grinblat, N. Pour and Z. Malik, Regulation of porphyrin synthesis and photodynamic therapy in heavy metal intoxication, J. Environ. Pathol., Toxicol. Oncol., 2006, 25, 145–158.
N. Grunberg-Etkovitz, L. Greenbaum, B. Grinblat and Z. Malik, Proteasomal degradation regulates expression of porphobilinogen deaminase (PBGD) mutants of acute intermittent porphyria, Biochim. Biophys. Acta, Mol. Basis Dis., 2006, 1762, 819–827.
G. G. Guo, M. Gu and J. D. Etlinger, 240-kDa proteasome inhibitor (CF-2) is identical toδaminolevulinic acid dehydratase, J. Biol. Chem., 1994, 269, 12399–12402.
M. Hrdinka, H. Puy and P. Martasek, May 2006 update in porphobilinogen deaminase gene polymorphisms and mutations causing acute intermittent porphyria: comparison with the situation in Slavic population, Physiol. Res., 2006, 55(Suppl 2), S119–S136.
S. D. Ickowicz, Y. Gozlan, L. Greenbaum, T. Babushkina, D. J. Katcoff and Z. Malik, Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma, Br. J. Cancer, 2004, 90, 1833–1841.
E. K. Jaffe and L. Stith, ALAD porphyria is a conformational disease, Am. J. Hum. Genet., 2007, 80, 329–337.
R. Kauppinen, Porphyrias, Lancet, 2005, 365, 241–252.
A. Klein, P. Babilas, S. Karrer, M. Landthaler and R. M. Szeimies, Photodynamic therapy in dermatology–an update 2008, J. Dtsch. Dermatol. Ges., 2008, 6, 839–846.
P. N. Liubchenko, B. N. Gladyshev and V. F. Mostun, Porphobilinogen biosynthesis fromδaminolevulinic acid by the internal organs of white rats, Biul. Eksp. Biol. Med., 1978, 86, 687–689.
M. Miyake, M. Ishii, K. Kawashima, T. Kodama, K. Sugano, K. Fujimoto and Y. Hirao, siRNA-mediated Knockdown of the Heme Synthesis and Degradation Pathways: Modulation of Treatment Effect of 5-Aminolevulinic Acid-based Photodynamic Therapy in Urothelial Cancer Cell Lines, Photochem. Photobiol., 2009, 85, 1020–1027.
N. M. Navone, C. F. Polo, A. L. Frisardi and A. M. Batlle, Mouse mammary carcinoma δ-aminolevulinate dehydratase, Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol., 1990, 96, 729–731.
P. Ponka, Tissue-specific regulation of iron metabolism and heme synthesis: distinct control mechanisms in erythroid cells, Blood, 1997, 89, 1–25.
P. Ponka and H. M. Schulman, Regulation of heme biosynthesis: distinct regulatory features in erythroid cells, Stem Cells, 1993, 11(s1), 24–35.
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Feuerstein, T., Schauder, A. & Malik, Z. Silencing of ALA dehydratase affects ALA-photodynamic therapy efficacy in K562 erythroleukemic cells. Photochem Photobiol Sci 8, 1461–1466 (2009). https://doi.org/10.1039/b9pp00007k
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DOI: https://doi.org/10.1039/b9pp00007k