A regulatory role for porphobilinogen deaminase (PBGD) in δ-aminolaevulinic acid (δ-ALA)-induced photosensitization?

Abstract

Summary As an initial approach to optimize 8-aminolaevulinic acid (6-ALA)-induced photosensitization of tumours, we examined the response of three enzymes of the haem biosynthetic pathway: 8-ALA dehydratase, porphobilinogen deaminase (PBGD) and ferrochelatase. Only PBGD activity displayed a time-and dose-related increase in tumours after intravenous administration of 300 mg kg-1 6-ALA. The time course for porphyrin fluorescence changes, reflecting increased production of the penultimate porphyrin, protoporphyrin IX (PPIX), showed a similar pattern to PBGD. This apparent correlation between PBGD activity and porphyrin fluorescence was also observed in four cultured tumour cell lines exposed to 0.1-2.0 mm 6-ALA in vitro. The increase in PBGD activity and PPIX fluorescence was prevented by the protein synthesis inhibitor cycloheximide. As the apparent Km for PBGD was similar before and after 6-ALA, the increase in PBGD activity was attributed to induction of enzyme de novo. These observations of an associated response of PBGD and PPIX imply that PBGD may be a ratelimiting determinant for the efficacy of 6-ALA-induced photosensitization when used in photodynamic therapy.Keywords: 6-aminolaevulinic acid: photosensitization; porphobilinogen deaminase: haem biosynthesis; porphyrin fluorescence Haem is an essential prosthetic group in many critical cellular proteins such as haemoglobin, cytochrome P450 and cyclooxygenase (Abraham, 1991). Eight enzymes are involved in the biosynthesis of haem, a process that occurs in two subcellular compartments: the mitochondria and the cytosol. The first enzyme in the haem pathway, mitochondrial 8-aminolaevulinic acid synthase (8-ALA-S), forms 6-ALA from glycine and succinyl CoA and is a target for the regulation of haem biosynthesis. Feedback inhibition of 8-ALA-S occurs when intracellular haem is present in excess (Ade, 1990).The last step in the haem biosynthetic pathway, the insertion of iron into PPIX to form haem, is catalysed by the mitochondrial enzyme ferrochelatase (FC). In unperturbed systems, FC is regulated by the availability of iron and/or PPIX. Two of the metabolic events that occur between 8-ALA-S and FC are catalysed by the cytosolic enzymes 8-ALA dehydratase (8-ALA-D) and porphobilinogen deaminase (PBGD). The dehydratase enzyme catalyses the condensation of two 8-ALA molecules to form porphobilinogen (PBG). It is the first enzyme that will metabolize the administered 8-ALA. The next enzyme in haem biosynthesis, PBGD, forms the tetrapyrole ring from four porphobilinogen molecules (Abraham, 1991).By providing the 8-ALA-S product, 8-ALA, the initial feedback step has been circumvented and this approach has been exploited for use in photodynamic therapy (PDT) of cancer (Kennedy and Pottier, 1992;Grant et al, 1993; Cairnduff et al, 1994;Regula et al, 1995). Traditional PDT regimens consist of the systemic adminis- Accepted 13 June 1997 Correspondence to: R Hilf tration of a photosensitizer followed by an appropriate interval to allow for its maximal accumulation in ma...

δ-Aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro

“…The data also show that both PBGD and PPIX levels were reduced by δ-ALAinduced photosensitization using either Experiment 3 or Experiment 5 conditions. These results lend support to the hypothesis that PBGD is a most important enzyme target when δ-ALA is administered exogenously (Gibson et al, 1998).…”

“…It is inferred that the second slowest step in the haem biosynthetic pathway, porphobilinogen deaminase (PBGD) (Healey et al, 1981;Ades, 1990), would be the next rate-limiting step after δ-ALA-S is circumvented. In a recent report, we demonstrated a relationship between PBGD activity and the amount of PPIX accumulated, both of which increased in the δ-Aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro presence of exogenous δ-ALA (Gibson et al, 1998). Presently, we extend those findings by examining the effect photosensitization would have on PBGD activity and PPIX accumulation.…”

Aminolaevulinic acid-induced photodynamic therapy: cellular responses to glucose starvation

“…The rate at which the cells generated PpIX (total production, i.e. intracellular and medium), was actually increased by chronic glucose deprivation, suggesting either induction of the rate limiting enzyme, thought to be porphobilinogen deaminase (Gibson et al, 1998), or a reduction in the rate of conjugation to iron to form haem by the enzyme ferrochelatase. There was a significant increase in the amount of PpIX in the medium, which may reflect increased generation, but it is also possible that the rate of PpIX leakage from the cells was increased.…”

Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma

“…Porphobilinogen deaminase (PBGD) then combines four molecules of PBG into hydroxymethylbilane, which is turned into uroporphyrinogen III. Uroporphyrinogen III converts to coproporphyrinogen and then, after reuptake in mitochondria, to PpIX (Szeimies et al, 1996;Ponka, 1997;Gibson et al, 1998). Fluorescence microscopy data suggest that because of the localisation of PpIX in the mitochondria, the primary cause of cell death after PDT is mitochondrial phototoxicity (Iinuma et al, 1994).…”

“…Aminolevulinic acid treatment of B16 melanoma cells induces PpIX biosynthesis and facilitates the photodynamic killing of these cells De Rosa and Bentley, 2000;Juzenas et al, 2002). Several mechanisms have been suggested as being involved in the photodynamic process, including a lower intracellular iron concentration, lowered ferrochelatase activity, higher permeability of ALA into tumours, higher local temperature and higher PBGD activity in malignant cells (el-Sharabasy et al, 1992;Ackermann et al, 1998;Gibson et al, 1998).…”