Is delta-aminolevulinic acid dehydratase rate limiting in heme biosynthesis following exposure of cells to delta-aminolevulinic acid?

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.

Relationship of delta-aminolevulinic acid-induced protoporphyrin IX levels to mitochondrial content in neoplastic cells in vitro.

Protoporphyrin IX, induced by the exogenous addition of delta-aminolevulinic acid, reaches different levels in different tumor cells. Because many of the steps in heme biosynthesis, of which protoporphyrin IX is penultimate, are located in the mitochondria, we surmised that the mitochondrial content of cells may relate to the amount of protoporphyrin IX synthesized in response to excess delta-aminolevulinic acid. We observed that accumulation of MitoTracker, a fluorescent mitochondrial probe, delta-aminolevulinic acid-induced protoporphyrin IX levels, and porphobilinogen deaminase activity all presented with the same cell-line-dependent rank order among the four different neoplastic cells. This rank order, however, differed for cytochrome c oxidase activity, the final enzyme in mitochondrial electron transport, and for accumulation of radioactive label from [(14)C]delta-aminolevulinic acid. The data demonstrate that enzymes involved in heme biosynthesis, in general, display a rank order associated with mitochondrial content. These data imply that such parameters may have value as prognosticators of cells to produce delta-aminolevulinic acid-induced protoporphyrin IX, a photosensitizer for photodynamic therapy of cancer.

Effect of estrogenic perturbations on delta-aminolevulinic acid-induced porphobilinogen deaminase and protoporphyrin IX levels in rat Harderian glands, liver, and R3230AC tumors.

The Harderian gland in rodents highly expresses enzymes of the heme biosynthetic pathway that are responsible for porphyrin production. Interestingly, many of the steps in Harderian gland heme biosynthesis, including protoporphyrin production, are controlled hormonally. We hypothesized that estrogenic alterations, ovariectomy or tamoxifen administration, might also alter the response of porphobilinogen deaminase activity and/or protoporphyrin IX production to delta-aminolevulinic acid administration in the hormonally responsive R3230AC rat mammary adenocarcinoma. We also determined whether the response of the R3230AC tumor, borne on ovariectomized hosts, to delta-aminolevulinic acid-based photodynamic therapy was altered compared with tumors treated on intact hosts. Ovariectomy of female Fischer rats bearing the hormonally responsive R3230AC mammary adenocarcinoma caused a significant reduction in delta-aminolevulinic acid-induced protoporphyrin IX levels and porphobilinogen deaminase activity in tumors compared with levels in tumors from intact animals treated with delta-aminolevulinic acid. In contrast, although porphobilinogen deaminase activity in the Harderian gland from ovariectomized animals was reduced significantly compared with that in glands from intact animals, protoporphyrin IX levels were unaltered. Administration of the anti-estrogen tamoxifen to tumor-bearing rats resulted in a significant increase in porphobilinogen deaminase in both tumor and Harderian gland. Although administration of delta-aminolevulinic acid increased protoporphyrin IX levels in Harderian glands in tamoxifen-treated animals, tumor levels of protoporphyrin IX remained unaltered in the tamoxifen-treated rats. Treatment of R3230AC tumors with delta-aminolevulinic acid-based photodynamic therapy in ovariectomized rats resulted in a significantly reduced response compared with the same treatment regimen in intact animals, 4.9+/-0.39 versus 10.6+/-0.6 days to reach twice the initial tumor volume, respectively. These results indicate that the hormonal status of the host should be considered when treating hormonally sensitive tumors with delta-aminolevulinic acid-based photodynamic therapy.

Effect of delta-aminolevulinic acid on protoporphyrin IX accumulation in tumor cells transfected with plasmids containing porphobilinogen deaminase DNA.

Recently, we reported that the delta-aminolevulinic acid (delta-ALA)-induced increase in porphobilinogen deaminase (PBGD) activity was closely correlated with an increase in the accumulation of protoporphyrin IX (PPIX), resulting in augmented phototoxicity. In this report, we asked whether increasing the cellular expression of PBGD by use of gene transfection techniques in vitro would further enhance delta-ALA-induced PPIX accumulation and hence, phototoxicity. For these experiments we constructed plasmid vectors containing the PBGD-DNA, using a reverse transcription-polymerase chain reaction-generated cDNA fragment encoded from its published sequence. Subsequently, transfection of the human mammary tumor cell line, MCF-7, and the human mesothelioma cell line, H-MESO-1, with the PBGD-DNA-containing plasmids was shown to produce a 2.5-2.7-fold increase in enzyme activity. Twenty-four hours after completion of the transfection procedure, transfectants were exposed for 3 h to 0.5 mM delta-ALA. Exposure of either wild type or transfectants to delta-ALA led to measurable levels of PPIX. Although this produced a modest but significant increase in intracellular PPIX content in H-MESO-1 cells compared to wild-type cells incubated with delta-ALA alone, the increase above the transfection control did not reach statistical significance. Likewise, a significant increase in PPIX was not observed in transfected MCF-7 cells subsequently exposed to delta-ALA. These data demonstrate that transient transfection of cells with the cDNA of PBGD was successful in elevating enzyme activity in both tumor cell lines, but this did not result in a comparable difference in the levels of PPIX. Such an approach for the study of other enzymes in the heme pathway should provide a model to better define rate-limiting steps in the delta-ALA induction of PPIX, and ultimately, to enhance the effectiveness of photodynamic therapy.

Delta-aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro.

Recently, considerable interest has been given to photodynamic therapy of cancer using delta-aminolaevulinic acid to induce protoporphyrin IX as the cell photosensitizer. One advantage of this modality is that protoporphyrin IX is cleared from tissue within 24 h after delta-aminolaevulinic acid administration. This could allow for multiple treatment regimens because of little concern regarding the accumulation of the photosensitizer in normal tissues. However, the haem biosynthetic pathway would have to be fully functional after the first course of therapy to allow for subsequent treatments. Photosensitization of cultured R3230AC rat mammary adenocarcinoma cells with delta-aminolaevulinic acid-induced protoporphyrin IX resulted in the inhibition of porphobilinogen deaminase, an enzyme in the haem biosynthetic pathway, and a concomitant decrease in protoporphyrin IX levels. Cultured R3230AC cells exposed to 0.5 mM delta-aminolaevulinic acid for 27 h accumulated 6.07 x 10(-16) mol of protoporphyrin IX per cell and had a porphobilinogen deaminase activity of 0.046 fmol uroporphyrin per 30 min per cell. Cells cultured under the same incubation conditions but exposed to 30 mJ cm(-2) irradiation after a 3-h incubation with delta-aminolaevulinic acid showed a significant reduction in protoporphyrin IX, 2.28 x 10(-16) mol per cell, and an 80% reduction in porphobilinogen deaminase activity to 0.0088 fmol uroporphyrin per 30 min per cell. Similar effects were evident in irradiated cells incubated with delta-aminolaevulinic acid immediately after, or following a 24 h interval, post-irradiation. There was little gain in efficacy from a second treatment regimen applied within 24 h of the initial treatment, probably a result of initial metabolic damage leading to reduced levels of protoporphyrin IX. These findings suggest that a correlation may exist between the delta-aminolaevulinic acid induction of porphobilinogen deaminase activity and the increase in intracellular protoporphyrin IX accumulation.

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

As an initial approach to optimize delta-aminolaevulinic acid (delta-ALA)-induced photosensitization of tumours, we examined the response of three enzymes of the haem biosynthetic pathway: delta-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) delta-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 delta-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 delta-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 rate-limiting determinant for the efficacy of delta-ALA-induced photosensitization when used in photodynamic therapy.

Time-dependent intracellular accumulation of delta-aminolevulinic acid, induction of porphyrin synthesis and subsequent phototoxicity.

Photodynamic therapy (PDT), a novel treatment for a variety of human malignancies, usually consists of visible light irradiation of lesions following the systemic administration of a photosensitizer. Induction of the endogenous photosensitizer protoporphyrin IX by the systemic or topical administration of delta-aminolevulinic acid (delta-ALA) is being investigated for use in PDT. We have determined that the incubation of two human and two rodent tumor cell lines in culture with delta-ALA over a 24 h period results in an increase in the accumulation of fluorescent porphyrins in all of these cell lines. However, the two human cell lines produce fluorescent porphyrin at different rates from those seen in the rodent cell lines. The uptake of 14C-delta-ALA was concentration dependent, similar for all the cell lines studied and rapidly reached an intra/extracellular equilibrium after delta-ALA was added to the culture medium. The increase in intracellular fluorescent porphyrin was dependent on the level of delta-ALA in the medium and the incubation time and was directly related to the phototoxicity observed upon exposure of cultured monolayers to light. The data demonstrate that equivalent levels of phototoxicity can be attained by exposing cells to 0.04 mM delta-ALA for 24 h or to 0.5 mM delta-ALA for 2 h. These findings may have implications for optimization of PDT treatment regimens that use delta-ALA.