Is δ-Aminolevulinic Acid Dehydratase Rate Limiting in Heme Biosynthesis Following Exposure of Cells to δ-Aminolevulinic Acid?¶

Abstract

Understanding the regulation and control of heme/porphyrin biosynthesis is critical for the optimization of the delta-aminolevulinic-acid (ALA)-mediated photodynamic therapy of cancer, in which endogenously produced protoporphyrin IX (PPIX) is the photosensitizer. The human breast cancer cell line MCF-7, the rat mammary adenocarcinoma cell line R3230AC, the mouse mammary tumor cell line EMT-6 and the human mesothelioma cell line H-MESO-1 were used to study ALA-induced PPIX levels and their relationship to delta-aminolevulinic acid dehydratase (ALA-D) activity in vitro. Incubation of these cell lines with 0.5 mM ALA for 3 h resulted in a significant increase in PPIX accumulation, compared with control cells, but there was no significant change in ALA-D activity. Exposure of cells incubated with ALA to 30 mJ/cm2 of fluorescent light, a dose that would cause a 50% reduction in cell proliferation, did not significantly alter the activity of ALA-D. Increasing the activity of porphobilinogen deaminase (PBGD), the enzyme immediately subsequent to ALA-D, by four- to seven-fold via transfection of cells with PBGD complementary DNA did not alter the activity of ALA-D. However, incubation of cells with various concentrations of succinyl acetone, a potent inhibitor of ALA-D, caused a concomitant decline in both PPIX accumulation and ALA-D activity. These data imply that when cells are exposed to exogenous ALA, ALA-D is an important early-control step in heme/porphyrin biosynthesis and that regulation of PPIX synthesis by this dehydratase may impact the effectiveness of ALA-mediated photosensitization.

Mechanistic studies on δ-aminolevulinic acid uptake and efflux in a mammary adenocarcinoma cell line

“…We found that neither ALA nor porphyrin pools regulate ALA incorporation, since the initial ALA uptake rates remain constant. In addition, the presence of SA during the whole assay without changing ALA uptake demonstrates that SA does not compete for ALA uptake (Figure 1), in good agreement with the findings of Gibson et al (2001).…”

“…Some haem enzymes such as ALA dehydratase and PBG deaminase have been postulated to be rate limiting in the production of porphyrins from ALA (Gibson et al, 1998(Gibson et al, , 2001). However, under our conditions of short ALA exposure, ALA availability is limiting tetrapyrrole formation.…”

Preclinical Characterization of 5-Amino-4-Oxo-[6-11C]Hexanoic Acid as an Imaging Probe to Estimate Protoporphyrin IX Accumulation Induced by Exogenous Aminolevulinic Acid

“…In the present study, 11 C-MALA uptake in tumor cells was strongly correlated with ALAD expression levels and ALA-induced PpIX accumulation. Because ALAD plays a rate-limiting role in regulating PpIX accumulation (19,20), 11 C-MALA would reflect PpIX accumulation in tumor cells as a sum of the ALA influx and metabolism by ALAD.…”

“…Therefore, a new noninvasive imaging method based on the mechanism of PpIX accumulation is needed. PpIX accumulation has been considered to be modulated by the influx of ALA via transporters (10,18) and the activity of enzymes that catalyze the biosynthesis of heme from ALA (19)(20)(21). PET enables functional and quantitative assessments of several biologic processes such as transport and metabolism (22,23).…”

“…The influx of ALA into cells (10,18) and expression levels of ALA dehydratase (ALAD) that catalyzes the first step of PpIX synthesis from ALA play a major and rate-determining role in regulating PpIX accumulation (19,20). Accordingly, a PET tracer to target ALA influx and ALAD expression would be useful for predicting PpIX accumulation in tumors.…”